KR102563030B1 - Recombinant cell surface capture proteins - Google Patents

Abstract

Recombinant cell surface capture proteins and detection molecules useful for isolating and detecting cells producing a secreted heterodimeric protein of interest (POI) having immunoglobulin CH3 domains and/or substituted CH3 domains are provided. Recombinant cell surface capture proteins and detection molecules that isolate and detect bispecific antibodies are also provided. The present invention also relates to recombinant antigens capable of recognizing and binding to a desired protein containing a CH3 domain and/or a modified CH3 domain, eg, a CH3 domain with or without amino acid substitutions in H95 and Y96 (IMGT)- binding proteins.

Description

Recombinant cell surface capture proteins {RECOMBINANT CELL SURFACE CAPTURE PROTEINS}

Cross-reference to related applications

This application claims the benefit under 35 USC § 119(e) of US Provisional Patent Application Serial No. 61/726,040, filed on November 14, 2012, which application is expressly incorporated herein by reference in its entirety. .

sequence listing

This application contains a sequence listing filed in computer readable form as file 8600WO_ST25.txt (86,267 bytes) created on November 12, 2013 by reference.

Field of the Invention

The field of the present invention relates to methods for identifying, isolating and enriching cells that produce recombinant cell surface capture proteins and secreted proteins that are heterodimers, eg, dual specific proteins. More specifically, cell surface capture proteins and methods provide rapid and efficient detection of high-expressing recombinant antibody-producing cell lines, including cells secreting specific hybridoma and heterodimeric proteins, e.g., bispecific antibodies. Allows separation, thereby enriching heterodimeric species (dual specific molecules) and preferentially separating heterodimeric species from homodimeric species.

Prior methods for expressing a gene of interest (GOI) in a host cell are known. Briefly, an expression vector carrying a GOI is introduced into a cell. Following stable integration, standard methods for isolating high-expressing cells are collection of cell pools, hand-picking of colonies from plates, limited dilution, or single cell counting by other methods known in the art. entails separation. The pool or individual clones are then expanded and screened for production of a protein of interest (POI) by direct measurement of POI activity, by immunological detection of the POI, or by other suitable techniques. These procedures are laborious, inefficient, and expensive, and the number of clones that can be analyzed is usually limited to a few hundred.

Most heterogeneity in protein expression by cells after stable integration necessitates that many individual clones be screened in an effort to identify rare integration events that give rise to stable, high-expressing production cell lines. This need requires methods that allow rapid identification and isolation of cells expressing the highest levels of protein production. In addition, harvesting clonal pools or hand-picked colonies carries the risk of losing high-expressing cells, which are often slower growing compared to faster-growing low-expressing cells. Therefore, there is a need to exist methods that allow rapid screening and isolation of individual cells that allow for high-level expression of secreted POIs. When a POI contains more than one subunit, it is necessary to preferentially select for the desired heterodimeric species over the homodimeric species.

The incorporation of flow cytometry into methods used for the isolation of stable expressing cell lines has improved the ability to screen large numbers of individual clones, but currently available methods are still insufficient for a variety of reasons. Diffusion of POIs among cells of different characteristics was also problematic.

A high-throughput screening method for rapid isolation of the cells secreting a protein by direct screening for a protein of interest (POI) is described. The present invention also allows convenient monitoring of POI expression on a single cell basis during the manufacturing process. Moreover, this technique can be directly applied to the screening of bispecific antibody-producing cells, or any cell that produces heterodimeric proteins. The technique can also be applied directly to the screening of cells that produce a modified T cell receptor, eg, cells that produce a soluble form of the T cell receptor.

In one aspect, the present invention provides a method for detecting and isolating cells that produce a secreted protein of interest (POI) comprising the steps of a) constructing a nucleic acid molecule encoding a cell surface capture molecule capable of binding to the POI; b) transfecting cells expressing the POI with the nucleic acid molecule of step a); c) detecting the surface-displayed POI by contacting the cell with a detection molecule, wherein the detection molecule binds to the POI; and d) isolating cells based on the detection molecule.

In various embodiments, the desired protein comprises a ligand, soluble receptor protein, growth factor, fusion protein, antibody, bispecific antibody, Fab, single chain antibody (ScFv), or fragment thereof. When the desired protein is an antibody, the antibody is selected from the group consisting of IgM, IgG, IgA, IgD or IgE, as well as various subtypes or variants thereof. In certain embodiments, the antibody is an anti-DII4 antibody, an anti-ErbB3 antibody, an anti-EGFR antibody, a bi-specific anti-ErbB3/EGFR bi-specific antibody, or an anti-IL-6 receptor antibody.

In a more specific embodiment, the protein of interest is interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-13, IL-13 15, IL-16, IL-17, IL-18, IL-21, ciliary neurotrophic factor (CNTF), erythropoietin, vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang-1), angi a growth factor selected from the group consisting of opoietin 2 (Ang-2), TNF, interferon-gamma, GM-CSF, TGFp, and TNF receptor.

In various embodiments, the protein of interest comprises a variable domain of a T cell receptor. In certain embodiments, the protein of interest is a soluble T cell receptor (sTCR), or a protein comprising a T cell receptor extracellular domain fused to Fc (TCR-Fc). In certain embodiments, the Fc is a human Fc. In various embodiments, the protein comprises a variable domain of a T cell receptor extracellular domain. In various embodiments, the protein comprises the variable and constant domains of a T cell receptor extracellular domain.

Nucleic acids encoding the desired proteins may be from any source, either naturally occurring or constructed through recombinant techniques, or may be selected from a DNA library.

In various embodiments, the cell surface capture molecule is a ligand-specific receptor, a receptor-specific ligand, an antibody-binding protein, an antibody or an antibody fragment such as a ScFv, or a peptide. When the capture molecule is a peptide, the peptide may be isolated from a phage display library. In a more specific embodiment, the capture molecule is Ang1, Ang2, VEGF, Tie1, Tie2, VEGFRI (Flt1), VEGFRII (Flk1 or KDR), CNTF, CNTFR-α, a cytokine receptor component, two or more cytokine receptor components It may be a fusion of or a fragment thereof. When the capture molecule is an antibody-binding protein, the antibody-binding protein is an Fc receptor, an anti-immunoglobulin antibody, an anti-immunoglobulin (anti-Ig) ScFv, an anti-Fc antibody, an anti-Fc* antibody, protein A, a protein L, protein G, protein H or functional fragments thereof. As such, in some embodiments, the capture molecule is a fusion protein comprising an antigen, protein A, or an anti-Ig ScFv fused to a transmembrane domain or GPI binder.

In various embodiments, where the protein of interest comprises a T cell receptor variable domain, the cell surface capture molecule comprises a membrane-bound antigen that can be recognized by an Fc receptor or a variable domain of a T cell receptor.

In some embodiments, where the desired protein is a heterodimeric protein, e.g., a heterodimeric protein having a first subunit and a second subunit, the cell surface capture molecule binds to the first subunit but not to the second subunit A capable antigen, protein A, or ScFv, or such a cell surface capture molecule binds to the second subunit but not to the first subunit.

In various embodiments, a bispecific antibody wherein the desired protein has one CH3 domain comprising IgG1, IgG2, IgG4, or a mutation that abolishes binding to Protein A, and the other CH3 domain capable of binding Protein A; or an Fc region of IgG1, IgG2, IgG4 or a fusion protein comprising an Fc region having one CH3 domain containing a mutation abolishing binding to Protein A and the other CH3 domain capable of binding to Protein A. , cell surface capture molecules include anti-immunoglobulin ScFvs, such as anti-Fc or anti-Fc* ScFvs.

In various embodiments, the method of the present invention provides for anchoring the POI to the cell membrane, and further comprises a membrane anchor that is exposed to the exterior of the cell and thus functions as a cell surface capture molecule. In certain embodiments, the membrane anchor is a transmembrane anchor or GPI link. Examples of specific transmembrane anchors include the transmembrane domain of an Fc receptor, eg, the transmembrane domain of human FcγRI, examples of which are cited in SEQ ID NO:17. Membrane anchors may be cell native, recombinant, or synthetic.

In various embodiments, the protein of interest comprises a T cell receptor variable region and the cell surface capture molecule comprises a membrane-bound antigen. In certain embodiments, a membrane-bound antigen is a recombinant fusion protein comprising an antigen recognizable by a T cell receptor variable region fused to a membrane anchor and the antigen bound to a cell surface. In certain embodiments, the recombinant fusion protein comprises an antigen fused to a transmembrane anchor or GPI link. In another specific embodiment, the cell surface capture molecule comprises a recombinant fusion protein comprising an antigen capable of binding a membrane anchor and a major histocompatibility (MHC) molecule, e.g., of a tumor antigen and a transformed phenotype. including but not limited to self proteins.

In a further embodiment, a signal sequence is added to the amino terminus of the POI, whereby the protein is transported to the cell surface and functions as a cell surface capture molecule. The signal sequence may be cell native, recombinant, or synthetic.

In various embodiments, blocking molecules that bind cell surface capture molecules are added from the expressing cell to neighboring cells to reduce the spread of the POI. In another embodiment, the diffusion of a POI from an expressing cell to surrounding cells and its attachment to said cells is reduced by increasing the viscosity of the medium.

Cells isolated by the method of the present invention may be antibody-producing cells fused to immortalized cells. In a more specific embodiment, the antibody-producing cell is a B-cell or a derivative thereof. B-cell derivatives may be plasma cells, hybridomas, myeloma, or recombinant cells.

In addition, the methods of the present invention are useful for the identification of B-cells and their derivatives, or hybridomas expressing secreted antibodies of a desired specificity, affinity or isotype. The present invention may also be used to isolate cells expressing desired levels of antibodies or antibody fragments.

Detection of cells with a displayed POI can be accomplished through the use of any molecule capable of directly or indirectly binding to the displayed POI. Such detection molecules may facilitate detection and/or isolation of cells displaying a POI. In one embodiment, two molecules that bind to each other and are labeled separately are used. Detection and/or separation may be accomplished through standard techniques known in the art.

In another aspect, the invention features a method for detecting and isolating cells that produce a secreted protein of interest (POI) comprising the steps of a) transfecting the cells with a nucleic acid encoding a cell surface capture molecule, the cell surface capture molecule can bind to the POI; b) simultaneously or sequentially transfecting the cells of a) with a second nucleic acid encoding the POI, wherein the POI is expressed and secreted; c) detecting the surface-displayed POI by contacting the cell with a detection molecule that binds the POI; and d) isolating the cells based on the detection molecule.

In another aspect, the invention features the steps of detecting and isolating cells producing a POI, comprising: a) detecting cells expressing a cell surface capture molecule in high yield; b) isolating and culturing the cells detected in (a); c) transfecting the cell of (b) with a nucleic acid encoding the POI, wherein the POI is secreted; d) detecting the surface-displayed POI by contacting the cell with a detection molecule that binds the POI; and e) isolating the cells based on the detection molecule.

In another aspect, the present invention provides a method for detecting and isolating cells that produce high levels of a protein of interest (POI), comprising: a) converting the cells to a nucleic acid encoding such a cell surface capture molecule capable of binding the POI; transfection, wherein the cells express the POI; b) detecting cells of (a) expressing the cell surface capture molecule in high yield; c) isolating and culturing high-yield cells; d) detecting the surface-displayed POI by contacting the cell with a detection molecule that binds the POI; and e) isolating the detected cells.

In another aspect, the invention provides a method for detecting and isolating cells that produce high levels of a heterodimeric protein, wherein (a) the cell comprises a membrane anchor domain and is capable of binding to the first subunit of the heterodimeric protein. transfection with a nucleic acid encoding a cell surface capture molecule, which is a fusion protein capable of expressing heterodimeric proteins; (b) detecting cells of (a) expressing the surface capture molecule in high yield; (c) isolating and culturing cells expressing the surface capture molecule in high yield; (d) detecting the heterodimeric protein on the surface of the isolated and cultured cells of step (c) with a detection molecule that binds to the second subunit of the heterodimeric protein; and (e) isolating the cells detected in step (d) that have the heterodimeric protein detected on their surface.

In another aspect, the invention provides a method for detecting and isolating cells that produce high levels of immunoglobulins, comprising: (a) transfecting the cells with a nucleic acid encoding a cell surface capture molecule capable of binding immunoglobulins; a step in which the cells express immunoglobulins; (b) detecting cells of (a) expressing the surface capture molecule in high yield; (c) isolating and culturing cells expressing the surface capture molecule in high yield; (d) detecting the immunoglobulin with a detection molecule that binds to the immunoglobulin on the surface of the isolated and cultured cells of step (c); and (e) isolating the cells detected in step (d) that have immunoglobulins detected on their surface.

In another aspect, the invention provides a method for detecting and isolating cells that produce high levels of a bispecific antibody, wherein (a) the cells are a fusion protein comprising a membrane anchor domain, such as a ScFv fusion protein, and a dual transfection with a nucleic acid encoding a cell surface capture molecule capable of binding to a specific antibody, wherein the cell expresses the dual specific antibody; (b) detecting cells of (a) expressing the surface capture molecule in high yield; (c) isolating and culturing cells expressing the surface capture molecule in high yield; (d) detecting the bispecific antibody with a detection molecule that binds to the bispecific antibody on the surface of the isolated and cultured cells of step (c); and (e) isolating the cells detected in step (d) that have the bispecific antibody on their surface.

In another aspect, a method of detecting cells producing a desired level of affinity drug comprising a T-cell receptor (TCR) variable region is provided.

In another aspect, a method for detecting a cell producing a desired level of TCR-Fc is provided, comprising (a) transfecting the cell with a nucleic acid encoding an Fc receptor capable of binding TCR-Fc; cells expressing antigens recognized by TCR-Fc; (b) detecting cells of (a) expressing TCR-Fc in high yield; (c) isolating and culturing cells expressing TCR-Fc in high yield; (d) detecting the antigen with a detection molecule on the surface of the isolated and cultured cells of step (c); and (e) isolating the cells detected in step (d) that have antigens detected on their surface.

In various embodiments, the TCR is selected from human TCRs and rodent TCRs such as rat, mouse, or hamster TCRs. In certain embodiments, the Fc is a human Fc. In another specific embodiment, the Fc is human Fc and the Fc receptor is a high affinity human Fc receptor. In certain embodiments, the high affinity human Fc receptor is human FcγRI.

In various embodiments, the cell surface capture protein is a surface-bound antigen. In certain embodiments, the antigen is bound to a surface by fusion to a transmembrane domain or GPI binder.

In some aspects of the method of selecting enriched cells producing a desired protein, the recombinant antigen-binding protein may be used as a cell surface capture protein (CSCP), detection molecule (DM), and/or blocking molecule. Therefore, the present invention provides recombinant antigen-binding proteins.

In one aspect, the invention provides a human IgG1-Fc domain, a human IgG2-Fc domain, or a human IgG4-Fc domain, or any protein comprising the amino acid sequence of SEQ ID NO:26, e.g., encoding a human Fc. A recombinant antigen-binding protein that binds to is provided. In some embodiments, the recombinant antigen-binding protein binds the polypeptide with a K _D of less than about 40 nM as measured in a surface plasmon resonance assay.

In some embodiments, the recombinant antigen-binding protein has a heavy chain variable region (HCVR) having an amino acid sequence at least 95% identical to SEQ ID NO:15, or a light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:16 (LCVR) of one or more complementarity determining regions (CDRs). In one case, the protein is heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO:27, HCDR-2 having the amino acid sequence of SEQ ID NO:28, amino acid sequence of SEQ ID NO:29 HCDR-3, light chain CDR-1 having the amino acid sequence of SEQ ID NO:30 (LCDR-1), and LCDR-2 having the amino acid sequence of SEQ ID NO:31. In some cases, the protein is a HCVR having an amino acid sequence at least 95% identical to SEQ ID NO:15 (some of which is identical to SEQ ID NO:15) and an amino acid sequence at least 95% identical to SEQ ID NO: 16 LCVRs (some of which are identical to SEQ ID NO:16).

Recombinant antigen-binding proteins are antibodies and are useful as detection molecules (DMs).

In some embodiments, the recombinant antigen-binding protein is a ScFv fusion protein, in some cases comprising a heavy chain variable domain having an amino acid sequence that is at least 95% identical (or identical) to SEQ ID NO:15, at least 95% to SEQ ID NO:16 a light chain variable domain having the same (or identical) amino acid sequence, and a membrane anchor domain. In one embodiment, the membrane anchor domain is from an Fc receptor, such as the transmembrane domain of the human FcγR1 protein, as shown by SEQ ID NO:17, or SEQ ID NO:21, which includes a transmembrane domain, as well as C -contains a terminal cytoplasmic domain (SEQ ID NO:18). In one specific embodiment, the ScFv fusion protein has the amino acid sequence of SEQ ID NO:19. It has a recombinant antigen-binding protein, which is a ScFv fusion protein, and is useful as CSCP and DM.

In another aspect, the invention provides a polynucleotide encoding the antigen-binding protein of the previous aspect. In one embodiment, as in the case where the antigen-binding protein is an antibody, the polynucleotide encodes a light chain. Similarly, a polynucleotide may encode a heavy chain. In cases where the antigen-binding protein is a ScFv fusion protein, the polynucleotide may encode the ScFv-FcγRTM-cyto fusion protein of SEQ ID NO: 19. For example, a polynucleotide of SEQ ID NO: 20 encodes SEQ ID NO: 19.

In another aspect, the present invention provides a nucleic acid vector comprising the polynucleotide of the previous aspect. In one embodiment, the vector comprises a polynucleotide operably linked to an upstream promoter and leading to a downstream polyadenylation sequence, which encodes an antigen-binding protein. The promoter can be any promoter, for example a CMV promoter. Thus, in one case, the vector may contain the sequence of SEQ ID NO:25. In one embodiment, the vector may contain a nucleic acid sequence encoding a selectable marker, such as for example neomycin resistance. In one embodiment, the vector may contain a nucleic acid sequence encoding an energy transfer protein, such as green fluorescent protein (GFP), or a derivative thereof, such as yellow fluorescent protein (YFP). Thus, in one case, the vector may contain the sequence of SEQ ID NO:24.

A vector may be circular or linear, episomal to the genome of a host cell, or integrated into the genome of a host cell. In some embodiments, the vector is a circular plasmid, which in one specific embodiment has the nucleic acid sequence of SEQ ID NO:23 for the ScFv-FcγR-TM-cyto-encoding polynucleotide, and in another specific embodiment an antibody heavy chain- A nucleic acid sequence of an encoding polynucleotide, and in another specific embodiment, an antibody light chain-encoding polynucleotide. In some embodiments, a vector is a linear construct, which may be integrated into a host cell chromosome. In one specific embodiment, the linear construct has the nucleic acid sequence of SEQ ID NO:22 for ScFv-FcγR-TM-cyto-encoding polynucleotide. In another specific embodiment, the linear construct comprises a nucleic acid sequence of an antibody heavy chain-encoding polynucleotide. In another specific embodiment, the linear construct comprises a nucleic acid sequence of an antibody light chain-encoding polynucleotide.

A host cell can be any cell, prokaryotic or eukaryotic. However, in one specific embodiment, the host cell is a CHO cell, eg a CHO-K1 cell.

In another aspect, the invention provides a host cell expressing the antigen-binding protein of the previous aspect and/or containing the polynucleotide or nucleic acid vector of the previous aspect. In some embodiments, the host cell is a CHO cell. In certain embodiments, the host cell is a CHO-K1 cell. In one embodiment, the host cell is used for production of the desired protein and the antigen-binding protein is used as a cell surface capture protein according to the methods disclosed herein.

In one aspect, the invention provides a host cell useful in the production of a protein of interest. The host cell has the polynucleotide or nucleic acid vector of the previous aspect and produces the antigen-binding protein of the previous aspect, which serves as a cell surface capture protein. Cell surface capture proteins bind to proteins of interest inside the host cell, are delivered through the cell's secretory machinery, and are expressed on the surface of the host cell. Thus, in one embodiment, the host cell comprises a cell surface capture protein located in the host cell plasma membrane, with the capturing moiety facing the exterior of the cell. In one embodiment, the cell surface capture molecule binds to a protein of interest, which is located on the plasma membrane and orients to the exterior of the cell.

In one embodiment, the host cell produces or can produce a ScFv fusion protein that binds to a desired protein containing an Fc domain, which contains a histidine at IMGT position 95 and a tyrosine at IMGT position 96. Examples include IgG1, IgG2, and IgG4 proteins. In one embodiment, the ScFv fusion protein contains the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In one specific embodiment, the ScFv fusion protein comprises the amino acid sequence of SEQ ID NO:19. In certain embodiments, the host cell binds to a bispecific antibody located at the plasma membrane and containing a heavy chain of at least one of IgG1, IgG2 or IgG4, or IgG1, IgG2 or IgG4, another type or containing one or more amino acid substitutions. cell surface capture proteins that may have a second heavy chain that

In one embodiment, the invention relates to (a) 95R, and (b) 95R and 96F according to the IMGT exon numbering system, or (a') 435R, and (b') 435R and 436F according to the EU numbering system. SEQ ID NO:42, which encodes a recombinant antigen-binding protein that binds to a substituted CH3 polypeptide comprising one or more amino acid substitutions selected from the group consisting of, or, for example, a substituted human Fc (also known as Fc*). Any protein comprising the amino acid sequence of In some embodiments, the recombinant antigen-binding protein binds the polypeptide with a K _D of less than about 60 nM as measured in a surface plasmon resonance assay.

In some embodiments, the recombinant antigen-binding protein has a heavy chain variable region (HCVR) having an amino acid sequence at least 95% identical to SEQ ID NO:38, or a light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:39 (LCVR) of one or more complementarity determining regions (CDRs). In one case, the protein comprises heavy chain CDR-1 (HCDR1) having the amino acid sequence of SEQ ID NO:32, HCDR-2 having the amino acid sequence of SEQ ID NO:33, HCDR-3 having the amino acid sequence of SEQ ID NO:34 , light chain CDR-1 (LCDR-1) having the amino acid sequence of SEQ ID NO:35, and LCDR-2 having the amino acid sequence of SEQ ID NO:36. In some cases, the protein is a HCVR having an amino acid sequence that is at least 95% identical to SEQ ID NO:38 (some of which is identical to SEQ ID NO:38) and an amino acid sequence that is at least 95% identical to SEQ ID NO:39. LCVRs (some of which are identical to SEQ ID NO:39).

In some embodiments, the recombinant antigen-binding protein is an antibody, which comprises a heavy chain and a light chain. The heavy chain may comprise an amino acid sequence that is at least 95% identical (or 100% identical) to SEQ ID NO:40. The light chain may comprise an amino acid sequence that is at least 95% identical (or 100% identical) to SEQ ID NO:41. Recombinant antigen-binding proteins that are antibodies are useful as detection molecules (DMs).

In some embodiments, the recombinant antigen-binding protein is a ScFv fusion protein, which in some cases comprises a heavy chain variable domain having an amino acid sequence that is at least 95% identical (or identical) to SEQ ID NO:38, and at least 95% identical to SEQ ID NO:39. light chain variable domains having % identical (or identical) amino acid sequences, and membrane anchor domains. In one embodiment, the membrane anchor domain is derived from an Fc receptor, such as the transmembrane domain of the human FcγR1 protein, as shown by SEQ ID NO:17, or SEQ ID NO:21, which includes a transmembrane domain, as well as but also the C-terminal cytoplasmic domain of SEQ ID NO:19. In one specific embodiment, the ScFv fusion protein has the amino acid sequence of SEQ ID NO:43. Recombinant antigen-binding proteins are ScFv fusion proteins and are useful as CSCPs and DMs.

In another aspect, the invention provides a polynucleotide encoding the antigen-binding protein of the previous aspect. In one embodiment, as in the case where the antigen-binding protein is an antibody, the polynucleotide encodes a light chain, eg, a light chain of SEQ ID NO:41. Similarly, the polynucleotide may encode a heavy chain, eg, a heavy chain of SEQ ID NO:40. In cases where the antigen-binding protein is a ScFv fusion protein, the polynucleotide may encode the ScFv-FcγR-TM-cyto fusion protein of SEQ ID NO:43. Representative example polynucleotides include those of SEQ ID NOs: 49, 50 and 51, respectively.

In another aspect, the present invention provides a nucleic acid vector comprising the polynucleotide of the previous aspect. In one embodiment, the vector comprises a polynucleotide operably linked to an upstream promoter and leading to a downstream polyadenylation sequence, which encodes an antigen-binding protein. The promoter can be any promoter, for example a CMV promoter. Thus, in one case, the vector may contain the sequence of SEQ ID NO:47. In one embodiment, the vector may contain a nucleic acid sequence encoding a selectable marker such as, for example, neomycin resistance. In one embodiment, the vector may contain a nucleic acid sequence encoding a green fluorescent energy transfer protein, such as green fluorescent protein (GFP), or a derivative thereof, such as yellow fluorescent protein (YFP). Thus, in one case, the vector may contain the sequence of SEQ ID NO:46.

Vectors may be circular or linear, episomal to the genome of the host cell, or integrated into the genome of the host cell. In some embodiments, the vector is a circular plasmid, which in one specific embodiment has the nucleic acid sequence of SEQ ID NO:44 for the ScFv-FcγR-TM-cyto-encoding polynucleotide, and in another specific embodiment an antibody heavy chain -has the nucleic acid sequence of an encoding polynucleotide, and in another particular embodiment has the nucleic acid sequence of an antibody light chain-encoding polynucleotide. In some embodiments, a vector is a linear construct, which may be integrated into a host cell chromosome. In one specific embodiment, the linear construct comprises the nucleic acid sequence of SEQ ID NO:51 for ScFv-FcγR-TM-cyto-encoding polynucleotide. In another specific embodiment, the linear construct comprises the nucleic acid sequence of SEQ ID NO:50 for an antibody heavy chain-encoding polynucleotide. In another specific embodiment, the linear construct comprises the nucleic acid sequence of SEQ ID NO:49 for an antibody light chain-encoding polynucleotide.

A host cell can be any cell, prokaryotic or eukaryotic. However, in one specific embodiment, the host cell is a CHO cell, eg a CHO-K1 cell.

In another aspect, the invention provides a host cell expressing the antigen-binding protein of the previous aspect and/or containing the polynucleotide or nucleic acid vector of the previous aspect. In some embodiments, the host cell is a CHO cell. In certain embodiments, the host cell is a CHO-K1 cell. In one embodiment, the host cell is used for production of the desired protein and the antigen-binding protein is used as a cell surface capture protein according to the methods disclosed herein.

In one aspect, the invention provides a host cell useful in the production of a protein of interest. The host cell has the polynucleotide or nucleic acid vector of the previous aspect and produces the antigen-binding protein of the previous aspect, which serves as a cell surface capture protein. Cell surface capture proteins bind to proteins of interest inside the host cell, are delivered through the cell's secretory machinery, and are expressed on the surface of the host cell. Thus, in one embodiment, the host cell comprises a cell surface capture protein located in the host cell plasma membrane, with the capturing moiety facing the exterior of the cell. In one embodiment, the cell surface capture molecule binds to a protein of interest, which is located on the plasma membrane and orients to the exterior of the cell.

In one embodiment, the host cell produces or can produce a ScFv fusion protein that binds to a desired protein containing an Fc domain, which contains an arginine at IMGT position 95 and a tyrosine at IMGT position 96 (Fc*). Examples include substituted CH3 regions of IgG3 and IgG1, IgG2, and IgG4 proteins. In one embodiment, the ScFv fusion protein contains the amino acid sequences set forth in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37 do. In one specific embodiment, the ScFv fusion protein comprises the amino acid sequence of SEQ ID NO:43. In certain embodiments, the host cell comprises a cell surface capture protein located at the plasma membrane and linked to IgG3 or substituted IgG1, IgG2 or IgG4, which contains arginine at IMGT position 95 and phenylalanine at IMGT position 96 (“Fc*”). , or bispecific antibodies containing at least one heavy chain of the Fc* type and the other heavy chain of the IgG1, IgG2 or IgG4 wild type.

In another aspect, the present invention provides methods for detecting, isolating, or enriching for cells stably expressing a protein of interest (POI). The method includes expressing a cell surface capture protein (CSCP) and a POI in a host cell. According to this method, CSCP binds to the "first site" on the POI to form a CSCP-POI complex inside the host cell. This CSCP-POI complex is then transported through the secretory system of the host cell and secreted from the cell. Since CSCP contains a membrane binding domain (eg, SEQ ID NO:17), the CSCP-POI complex is displayed on the surface of the host cell, and the POI is exposed outside the cell. Depending on the method, the host cell is then contacted with a detection molecule (DM), which binds to a "second site" on the POI. Those cells that bind DM are selected for identification, isolation, pooling, and/or enrichment. In one embodiment, DM-binding host cells are selected by fluorescence activated cell sorting.

In one embodiment, the method also includes contacting the cell with the blocking molecule prior to selecting the host cell. The blocking molecule binds to any CSCP that is not bound to the POI. Blocking molecules do not bind to the CSCP-POI complex.

In some embodiments, a POI contains multiple subunits, such as an antibody comprising two heavy chains and two light chains. In this case, the first portion on the POI may be on the first subunit, and the second portion on the POI may be on the second subunit. In some embodiments, a POI contains multiple subunits, such as heterodimeric proteins. In the case of heterodimeric proteins, the first site on the POI may be on a first subunit, such as a first receptor, and the second site on the POI may be on a second subunit, such as a second receptor or coreceptor. there is. In some embodiments, heterodimeric proteins are different receptors that interact to form heterodimers. When the POI is an antibody, the first site on the POI may be on a first heavy chain and the second site on the POI may be on a second heavy chain. In some embodiments, an antibody contains subunits that differ by at least one amino acid, such as antibodies having at least one heavy chain with a wild-type CH3 domain and another heavy chain with at least one amino acid substitution in the CH3 domain. In this case, the CSCP may be an antigen-binding protein, eg, an antigen or anti-Ig ScFv fusion protein, as described herein. As used herein, a detection molecule (DM) may include a recombinant antigen-binding protein labeled as described herein, eg, a labeled antigen or anti-Ig antibody or ScFv molecule.

In some cases, for example when the POI is a dual specific antibody, the first site contains a CH3 domain containing a histidine residue at position 95 according to the IMGT exon numbering system and a tyrosine residue at position 96 according to the IMGT exon numbering system. (Fc). Then the second site may be present on the heavy chain with a CH3 domain containing an arginine residue at position 95 according to the IMGT exon numbering system and a phenylalanine residue at position 96 according to the IMGT exon numbering system (Fc*). In this case, the CSCP is a ScFv fusion protein containing the amino acids of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, as in the previous aspect. may be an antigen-binding protein described; This includes SEQ ID NO:19 in certain embodiments. Also herein the detection molecule (DM) contains the amino acid sequences of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37 It may also include a labeled recombinant antigen-binding protein described in the previous aspect, such as an antibody or ScFv molecule that does; This includes SEQ ID NO:40 and SEQ ID NO:41 (anti-Fc* antibodies), or SEQ ID NO:43 (ScFv*) in certain embodiments. As used herein, a blocking molecule can be an Fc polypeptide (eg, single chain), such as hFc, or any molecule capable of binding to CSCP without binding to DM. In one embodiment, the detection molecule may be labeled anti-human IgG F(ab')2.

In case the POI is a dual specific antibody, the first site is on the heavy chain with a CH3 domain containing an arginine residue at position 95 according to the IMGT exon numbering system and a phenylalanine residue (Fc*) at position 96 according to the IMGT exon numbering system may be in Then the second site may be on the heavy chain with a CH3 domain containing a histidine residue at position 95 according to the IMGT exon numbering system and a tyrosine residue at position 96 according to the IMGT exon numbering system. In this case, the CSCP is a ScFv fusion containing the amino acid sequences of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37 Like a protein, it may be an antigen-binding protein described in the previous aspect; This includes SEQ ID NO:43 in certain embodiments. Also herein, a detection molecule (DM) is an antibody or ScFv molecule containing the amino acid sequences of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ NO:31 As in, may include a labeled recombinant antigen-binding protein described in the previous aspect, which in certain embodiments comprises a heavy chain and a light chain (anti-hFc antibody), or SEQ ID NO: 19 (ScFv). Here, the blocking molecule can be an Fc* polypeptide (eg single chain), or any molecule capable of binding to CSCP without binding to DM. In one embodiment, the detection molecule may be labeled anti-human IgG F(ab')2.

In some embodiments, the present invention provides a method for detecting or isolating cells stably expressing a heterodimeric protein comprising (a) expressing a cell surface capture protein (CSCP) and a heterodimeric protein in a host cell, (i) ) CSCP binds to a first site on the heterodimeric protein to form a CSCP-heterodimer protein complex inside the host cell, (ii) the CSCP-heterodimer protein complex is transported through the host cell, (iii) then the host cell Displayed on the surface of the; (b) contacting the host cell with a detection molecule, wherein the detection molecule binds to a second site on the heterodimeric protein; and (c) selecting a host cell that binds the detection molecule. In some embodiments, the heterodimeric protein comprises multiple subunits and the first portion on the heterodimeric protein is on the first subunit and the second portion on the heterodimeric protein is on the second subunit. In some embodiments, the cell surface capture molecule comprises an antigen, protein A, or ScFv capable of binding a first subunit but not a second subunit.

In one aspect, the invention provides a cell surface capture protein ("CSCP") in a host cell, an antibody light chain, a first antibody heavy chain containing a CH3 domain comprising a histidine at IMGT position 95 and a tyrosine at IMGT position 96, and an IMGT position expressing a second antibody heavy chain containing a CH3 domain comprising an arginine at 95 and a phenylalanine at IMGT position 96. Inside the host cell, CSCP binds to the first antibody heavy chain but not to the second antibody heavy chain, while the second antibody heavy chain binds to the first antibody heavy chain and the light chain to the heavy chain, thus the CSCP-antibody triple complex form This triple complex is secreted and presented onto the surface of the host cell. The host cell may also be contacted with a blocking molecule, which binds CSCP on the cell surface but only in those cases where CSCP does not bind the desired antibody, ie "empty" CSCP. The host cell is then contacted with a DM capable of binding or binding to a second antibody heavy chain. Host cells that bind DM are identified, selected, and/or pooled. In some embodiments, host cells that bind DM are selected, pooled, cultured and expanded, then subjected to another round of expression, detection, selection, pooling and expansion. This process may be repeated several times to enrich for the production of high titer bispecific antibodies.

In one embodiment, the CSCP used in the method is a ScFv-fusion containing the amino acid sequences of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31 It is protein. In one embodiment, CSCP comprises the amino acid sequence of SEQ ID NO:19. In one embodiment, the DM used in the method is the amino acid sequence of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37 is a protein containing In one embodiment, the DM is an antibody comprising the heavy chain sequence of SEQ ID NO:40 and the light chain sequence of SEQ ID NO:41. In another embodiment, the DM is a ScFv fusion protein containing the amino acid sequence of SEQ ID NO:43. A label, such as a fluorescent moiety like FITC or Alexa Fluor® 488, may also be attached to the DM. Fluorescence-activated cell sorting can also be used as a means of detection and selection.

In an alternative embodiment, the method of producing the bispecific antibody comprises a cell surface capture protein ("CSCP"), antibody light chain, CH3 comprising arginine at IMGT position 95 and phenylalanine 96 (Fc*) at IMGT position 96 (Fc*) in a host cell. expressing a first antibody heavy chain containing the domain, and a second antibody heavy chain containing a CH3 domain comprising a histidine at IMGT position 95 and a tyrosine at IMGT position 96. Within the host cell, CSCP binds to the first antibody heavy chain but not to the second antibody heavy chain, while the second antibody heavy chain binds to the first antibody heavy chain and the light chain to the heavy chain, thus the CSCP-antibody triple complex form This trimeric complex is secreted and presented onto the surface of host cells. The host cell may also be contacted with a blocking molecule, which binds CSCP on the cell surface but only in those cases where CSCP does not bind the desired antibody, ie "empty" CSCP. The host cell is then contacted with a DM that binds or is capable of binding to a second antibody heavy chain. Host cells that bind DM are identified, selected, and/or pooled. In some embodiments, host cells that bind DM are selected, pooled, cultured and expanded, then subjected to another round of expression, detection, selection, pooling and expansion. This process may be repeated several times to enrich for the production of high titer bispecific antibodies.

In one of these alternative embodiments, the CSCP used in the method is SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID It is a ScFv-fusion protein containing the amino acid sequence of NO:37. In one embodiment, CSCP comprises the amino acid sequence of SEQ ID NO:43. In one embodiment, the DM used in the method is a protein containing the amino acid sequences of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In one embodiment, the DM is an antibody comprising a heavy chain sequence and a light chain sequence. In another embodiment the DM is a ScFv fusion protein containing the amino acid sequence of SEQ ID NO: 19. A label, such as a fluorescent moiety like FITC or Alexa Fluor® 488, may also be attached to the DM. Fluorescence-activated cell sorting can also be used as a means of detection and selection.

In both the first and alternative embodiments, the host cells that are the product of iterative selection, pooling, and expansion are capable of, or produce, the bispecific antibody at a titer of at least 2 g/L, and the bispecific antibody is The antibody species (Fc/Fc*) represents at least 40% by mass (Fc/Fc + Fc Fc* + Fc/Fc*) of the total antibody produced by the host cell.

Other objects and advantages will become apparent from a review of the detailed description that follows.

Before the present methods are described, it should be understood that the methods and conditions may vary, as the present invention is not limited to the particular methods and experimental conditions described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting as the scope of the present invention will only be limited by the appended claims.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” refer to plural referents unless the context clearly dictates otherwise. includes Thus, for example, reference to “a method” includes one or more methods and/or steps of the type described herein and/or upon reading this disclosure or the like that will become apparent to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

general description

The methods of the present invention offer significant advantages over current methods for isolating and identifying protein-secreting cells. For example, cells secreting antibodies may be rapidly and conveniently isolated based on the desired specificity, affinity, or isotype. In addition, the amount of secreted protein produced can also be directly quantified unlike many methods in the prior art and the production of secreted protein is quantified indirectly.

Recently, two additional methods utilizing flow cytometry have been developed for high-throughput isolation of stable, high-expressing cell lines. The first method involves modification of the expression plasmid to contain private reads for the GOI mRNA. This is most often achieved by inserting, between the stop codon and the terminal poly A site of the GOI, an internal ribosome entry site (IRES) and a gene whose protein product is readily observed by flow cytometry, most frequently green fluorescent protein (GFP). (Meng et al. (2000) Gene 242:201). The presence of an IRES allows POI and GFP to be translated from the same mRNA. Therefore, the expression level of the GFP gene is indirectly related to the mRNA level for GOI. Clones that accumulate high levels of GFP are isolated by flow cytometry and then screened for POI production. As this method relies on the coupling of the reporter gene with GOI expression by the use of an IRES in the recombinant construct, it is not applicable to the isolation of hybridomas.

The use of flow cytometry for the isolation of expressing clones allows rapid analysis of large numbers of clones in a high-throughput, high-throughput format. Moreover, the use of flow cytometry greatly reduces the direct handling of cells. Unfortunately, the level of GFP production is not a direct measure of the level of POI production. A variety of mechanisms may separate the production of secreted POI from the accumulation of GFP. Differences in the production of POI and GFP reporters may be due to differences in translation efficiency of the two genes, secretion efficiency of POI, or stability of polycistronic mRNA.

Another method of using flow cytometry to isolate expressing clones involves encapsulation of cells in agarose microdroplets (Weaver et al. (1990) Methods Enzymol. 2:234). In this method, a biotinylated antibody specific for POI is coupled to biotinylated agarose via streptavidin, whereby the secreted POI is captured and retained within microdroplets (Gray et al., (1995) J (Immunol. Methods 182:155). A trapped POI is detected by immuno-staining with an antibody specific for the POI. To reduce the encapsulated agarose from absorptive POIs secreted from adjacent cells, the cells are placed in a low-permeability medium. The cells stained with the highest antibody of the POI in the embedding agarose are identified and isolated by flow cytometry. The gel microdroplet approach directly screens cells for their ability to secrete POI, instead of indirectly screening for expression of GOI mRNA, but requires the availability of antibodies suitable for trapping and staining the secreted POI and the process is agar Special equipment is needed to create the Ross Gel microdroplets. Moreover, some cells may be sensitive to the encapsulation process.

A variation of this method circumvents the requirement for embedding cells in a matrix by binding antibodies specific to the POI directly to the cell surface (Manz et al. (1995) PNAS 92:1921-1925). In this method, non-specific biotinylation of cell surface proteins with biotin-hydroxysuccinimide esters is followed by contact with a streptavidin-conjugated antibody capable of binding the POI. Cells secreting the POI were then coated with the POI, which was detected with an appropriately labeled secondary antibody. However, diffusion of POI between neighboring cells is problematic, and this method also requires a high viscosity medium to reduce the diffusion of POI. Since this highly viscous medium is necessary for identification of the cells, the cells should be washed and, if necessary, placed in a medium suitable for cell sorting.

The problems associated with the identification and isolation of high expressing recombinant cell lines apply particularly to the isolation of hybridomas expressing the desired antibody. However, identification of useful hybridomas involves several additional problems; They should be screened first for antigen-binding activity and then for immunoglobulin isotype. Moreover, GFP-based methods are not applicable for the identification and isolation of hybridomas because the constructs of hybridomas do not contain recombinant constructs, whereby the expression of antibody genes can be coupled to transcriptional reporters such as GFP. Hybridoma screening is a slow, laborious endeavor when the number of clones to be screened is limited by existing techniques.

The present invention describes a new and previously unknown method for identifying and isolating cells that produce secreted proteins. The present invention is based on the production of a cell line that expresses a molecule that is located on the cell surface and binds to the POI. Cell surface-displayed POIs can then be detected by labeling with various detection molecules. The amount of POI displayed on the cell surface is a direct measure of the total amount of POI secreted under specific conditions. POI producers may then be separated from non-producers, and production levels or POI characteristics may be differentiated. An advantage of the present invention is that it directly quantifies the secreted POI instead of an indirect measurement of mRNA.

The present invention relates to the use of constructs or cells that express cell surface capture molecules that bind to a variety of secreted POIs in the same cells that produce the POI. During cell secretion of POI, these cell surface capture molecules bind to it, or a complex of POI and cell surface capture molecule may be formed within the cell and then secreted. Binding may occur in an autocrine manner or during secretion. Cells producing the secreted POI may then be identified and isolated. Such identification and separation may be based on the nature of the POI, its production or lack thereof, or it may be by a specified level of production. The cell surface capture molecule and/or POI may be produced by the cell in its native state, or the cell surface capture molecule and/or POI may be produced recombinantly. Through the structure or use of these cells, any secreted protein can be captured by a given cell surface capture molecule, providing that there is a corresponding affinity between the two. As further described, any molecule can be engineered so that it can be used as a cell surface capture molecule. Therefore, the present invention may be utilized to isolate any cell secreting a protein.

Most proteins have the ability to function as cell surface capture molecules as described by the present invention. What is required is the ability of the desired protein to be anchored to the cell membrane and exposed to the extracellular space. If the desired cell has a signal sequence, but is not limited to, only a transmembrane anchor or a membrane anchor containing a GPI binding signal should be added to the cell surface capture molecule so that it remains anchored in the cell membrane exposed to the outside of the cell. . Additionally, if the desired protein lacks a signal sequence, the signal sequence may be added to the amino terminus of the desired protein, thereby delivering it to the cell surface. Signal sequences and membrane anchors may be cell native, recombinant, or synthetic.

Cells often secrete a variety of proteins either endogenously or after introduction of recombinant DNA. Any secreted protein can be identified and cells producing it can be isolated according to the method of the present invention. These secreted proteins include growth factors, growth factor receptors, ligands, soluble receptor components, antibodies, bispecific antibodies, recombinant trap molecules, Fc-containing fusion proteins, sTCRs, TCR-Fc, and peptide hormones. However, it is not limited thereto. These secreted proteins may or may not be recombinant. That is, secretion of some desired proteins from desired cells may not require the introduction of additional nucleotide sequences. For example, secretion of an antibody from a B-cell or plasma cell is not a result of introduction of recombinant nucleotide sequences into the B-cell or plasma cell. Secreted recombinant proteins may be produced by standard molecular biology techniques well known to those skilled in the art (see, e.g., Sambrook, J., E. F. Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1 , 2, and 3, 1989; Current Protocols in Molecular Biology, Eds. Ausubel et al., Greene Publ. Assoc., Wiley Interscience, NY). This secreted protein is useful for commercial and research purposes. The present invention includes the production of these secreted proteins through the methodology of the present invention. Detection of cells with a displayed POI can be accomplished through the use of any molecule capable of directly or indirectly binding to the displayed POI. Such detection molecules may facilitate detection and/or isolation of cells displaying a POI.

The present invention relates, inter alia, to a) ligand-producing cells that use ligand-specific receptors as cell surface capture molecules, b) soluble receptor-producing cells that use surface-bound receptor-specific ligands as cell surface capture molecules. , c) antibody-producing cells using antibody-binding proteins as cell surface capture molecules, d) using s-TCR-binding proteins (and, eg, antigens recognized by TCRs) as cell surface capture molecules. sTCR, e) TCR-Fc using an Fc-binding protein as a cell surface capture molecule, or f) using a fusion protein capture molecule comprising a ScFv domain fused to the FcγR transmembrane and cytoplasmic domains, abrogating Protein A binding It is applicable to the isolation of bispecific antibodies that have a mutation in one of the CH3 domains.

According to the methodology of the present invention, cells are first transfected with a vector containing a nucleotide sequence encoding a cell surface capture molecule capable of binding to a secreted POI, under conditions in which the cell surface capture molecule is expressed. Transfected cells that are appropriate producers of these cell surface capture molecules are then detected and isolated, and these cells are cultured. These cells may produce the POI naturally, or the POI may be produced recombinantly. When cells naturally produce POIs, they are ready to be detected and isolated. If the POI is recombinantly produced, then isolated and cultured cells expressing the specified cell surface capture molecule are transfected with a second nucleotide sequence encoding the POI, under conditions in which the secreted POI is expressed. Upon expression, the secreted POI binds to cell surface capture molecules and cells displaying the bound POI are detected and isolated.

If the POI is naturally produced by the cell, the cell will not be transfected with the nucleotide sequence encoding the POI. Therefore, this aspect of the present invention is applicable to all cells producing POIs. Moreover, where the cell surface capture molecule is naturally produced by the cell, the cell need not be transfected with a nucleotide sequence encoding the cell surface capture molecule. Therefore, this aspect of the present invention is applicable to all cells producing cell surface capture molecules.

A variety of host cells may be transfected. These cells may be of eukaryotic or prokaryotic origin. Cells are often immortalized eukaryotic cells, and in particular mammalian cells such as monkey kidney cells (COS), Chinese hamster ovary cells (CHO), HeLa cells, baby hamster kidney cells (BHK), human embryonic kidney cells ( HEK293), leukocytes, myeloma, cell lines transfected with adenovirus genes, including but not limited to immortalized human retinal cells transfected with adenoviral genes, such as PER.C6™ cells, e.g. , AD5 E1, and embryonic stem cells. Cells may also be non-mammalian cells, including bacterial, fungal, yeast and insect cells, such as Escherichia coli, Bacillus subtilus, Aspergillus species ( Aspergillus species), Saccharomyces cerevisiae, and Pichia pastoris, but are not limited thereto. All cells may be grown in culture tray media or in synergistic hosts under appropriate conditions. Most preferred cells will be culturable mammalian cells.

Secreted POIs bound to cell surface capture molecules may be detected and isolated by a variety of techniques known in the art. A cultured cell displaying a secreted POI may be contacted with (a) a molecule(s) capable of directly or indirectly binding to the secreted POI, such detection molecule(s) being, for example, chromogenic, fluorogenic. , a detection label such as a colored, fluorescent, or magnetic label. A label bound to the detection molecule may be detected and the cells may be isolated using a variety of methods. Most preferably, the label will be detected within the cell population and the cells will be isolated using flow cytometry. Alternatively, the detection molecule may be used for direct isolation of cells displaying a POI. This may be achieved by conjugation of the detection molecule to a culture plate, paramagnetic molecule, or some other particle or solid support. Moreover, the displayed POI may be directly detected by detection molecules or properties of the POI.

In one embodiment, two detection molecules that bind to each other and are labeled separately are used to detect the displayed secreted POI that blocks the interaction. When a cell displays a secreted POI that binds to the first detection molecule and blocks the interaction between the first and second detection molecules, the cell separates based on the presence of only the first detection molecule on its surface It could be. On the other hand, if a cell displays a secreted POI that binds to the first detection molecule but does not block the interaction between the first and second detection molecules, the cell is based on the presence of two detection molecules on its surface. may be separated by For example, antibody-producing cells expressing antibodies that specifically block or do not block the formation of receptor-ligand complexes may be identified. Where the detection molecules are separately labeled receptors and their ligands, then antibody-producing cells expressing antibodies that block receptor-ligand complexes from forming may be detected by the presence of one label on their surface. , antibody-producing cells expressing antibodies that do not block receptor-ligand complexes from forming may also be detected by the presence of two labels on their surface.

In any of the embodiments and with respect to separating expressing cells from non-expressing cells or less expressing cells, one of the main difficulties is the diffusion of the POI between neighboring cells when the POI is a secreted protein. Therefore, it is important for any system designed to capture secreted POI on the cell surface to prevent POI diffusion from expressing cells to surrounding cells and their attachment to those cells. If diffusion occurs and surrounding cells are coated with the secreted POI, segregation of cells based on the degree of POI application will fail to distinguish high-expressing cells from cells with low expression levels, and expressing cells from non-expressing cells. may fail to separate effectively from

Therefore, one embodiment of the present invention is to block the diffusion of secreted POI between neighboring cells. This may be achieved by the addition of a cell surface capture molecule or blocking molecule that binds to the POI and prevents binding of the secreted POI to the cell surface capture molecule. In this embodiment, the detection molecule does not bind to the blocking molecule. For example, when the cell surface receptor is hFcγRI and the secreted POI possesses a human IgG Fc fragment, then diffusion of the secreted POI between surrounding cells may be blocked by the addition of exogenous rat IgG to the culture medium. Display of the secreted POI and detection of cells that do not bind rat IgG is achieved by use of an antibody specific for human IgG Fc that does not recognize rat IgG. In another embodiment, binding of the secreted POI between surrounding cells is reduced by increasing the viscosity of the medium.

In one embodiment of the invention, the secreted POI cannot accumulate in the medium. This may be achieved by modulating the expression of the secreted POI and/or cell surface capture molecule such that short expression of the POI results in an amount of POI sufficient to bind to the cell surface capture molecule but insufficient for diffusion. In another embodiment, cells may be removed from the medium containing the accumulated POI, the POI bound to the cells is stripped, and POI expression continues for a limited period of time so that the secreted POI does not accumulate in the medium. Proteins may also be stripped by methods known in the art, for example by washing the cells with a low pH buffer.

According to the present invention, in a cell population that binds the majority of the detection molecule, said cells also express the majority of the secreted POI. In fact, the more POIs an individual cell secretes, the more POIs displayed on the cell surface. This correlation between the amount of surface-displayed POI and the expression level of the POI in the cell allows for rapid identification of cells with the desired relative expression level in a population of cells.

In one embodiment, DNA libraries may be used to express secreted proteins that may be displayed on the cell surface by cell surface capture molecules. For example, libraries of DNA may also be generated from coding regions of antibody variable domains of B-cells isolated from immunized animals. The DNA library may then be expressed in cells expressing cell surface capture molecules specific to the antibody such that clones of the desired specificity, isotype, or affinity may be identified and isolated by the methods of the present invention. In another embodiment, a library of DNA is generated from the coding region of the T cell receptor variable domain of a T-cell and may be fused to an Fc capable of binding, for example, an Fc-binding protein. The DNA library may then be expressed in cells expressing Fc-binding proteins such that clones of the desired specificity, isotype, or affinity may be identified and isolated as described herein.

In another embodiment, transgenic mammals may be created that express specific cell surface capture molecules in one or more cell types. Cells of such transgenic mammals may then be directly screened for production of POIs. For example, it may be desirable to express cell surface capture molecules specific to the antibody in plasma cells. Thus, plasma cells of immunized mice may be harvested and antibodies producing such antibodies specific for the desired antigen may be isolated by the method of the present invention.

In a further embodiment of the invention, antibody production is measured through the use of a CHO cell line expressing the human FcγR1 receptor (FcγRI) that binds to a specific antibody or POI, TCR-Fc.

In another aspect of the invention, the protein of interest comprises one or more T cell receptor variable domains or soluble T cell receptors. One or more T cell receptor variable domains may be covalently linked to a moiety capable of binding a cell surface capture protein. In certain embodiments, one or more T cell receptor variable domains are fused to an Fc sequence, eg a human Fc sequence, and the cell surface capture protein is an Fc receptor, eg FcγR.

The general structure of TCR variable domains is known (see, eg, Lefranc and Lefranc (2001) The T Cell Receptor FactsBook, Academic Press, incorporated herein by reference; eg, pp. 17-20; see also Lefranc et al. (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Developmental and Comparative Immunology 27:55-77, and Lefranc et al. see receptor constant domains and Ig superfamily C-like domains, Developmental and Comparative Immunology 29:185-203, each incorporated herein by reference). In one embodiment, the TCR variable domain of a TCR-Fc comprises an N-terminal region with a variable domain of 104-125 amino acids. In another embodiment, the TCR-Fc further comprises a TCR constant domain comprising amino acids 91-129. In another embodiment, the TCR-Fc further comprises a linking peptide comprising 21-62 amino acids.

In one embodiment, an Fc sequence is fused to a TCR variable domain either directly or via a linker. In another embodiment, a TCR-Fc comprises a TCR variable region and a TCR constant domain, and the Fc sequence is fused to the TCR constant domain either directly or via a linker. In another embodiment, a TCR-Fc comprises a TCR variable region, a TCR constant domain, and a linking peptide, and the Fc sequence is fused to the linking peptide either directly or through a linker.

An sTCR, TCR-Fc, or fusion protein comprising one or more T cell receptor variable regions may be a desired antigen, e.g., a substrate produced by tumor cells, e.g., a tumor cell substrate capable of producing an immune response in the host. can be selected to specifically bind to. In certain embodiments, an antigen is an antigen that is present on the surface of a tumor cell (ie, a tumor antigen), is recognized by a T cell, and produces an immune response in the host. Tumor antigens include, for example, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), MUC-1, epithelial tumor antigen (ETA), tyrosinase (eg for melanoma), melanoma-associated antigen (MAGE), and mutated or abnormal forms of other proteins such as ras, p53, and the like.

In one embodiment, the POI is TCR-Fc, wherein the TCR-Fc comprises a TCR α chain variable region fused to an Fc sequence and a TCR β chain (each directly or via a linker) fused to an Fc sequence, wherein the TCR α chain Chain-Fc fusions and TCR β chain-Fc fusions combine to form αβ TCR-Fc. In certain embodiments, an αβ TCR-Fc comprises two polypeptides: (1) a TCR α chain variable region fused to a TCR α chain constant domain fused to an Fc sequence, and (2) a TCR fused to an Fc sequence. TCR β chain variable region fused to β chain constant domain.

In another embodiment, the POI is a TCR-Fc having a TCR α variable region and a TCR β variable region and, optionally, a TCR α constant domain and/or a TCR β constant domain. In certain embodiments, a TCR-Fc comprises (5' to 3') a TCR α variable region sequence, then optionally a TCR α constant domain sequence, a TCR β variable region sequence, then optionally a TCR β constant domain sequence, optionally a binding Well, it is then encoded by a nucleic acid comprising an Fc sequence. In a specific embodiment, a TCR-Fc comprises (5' to 3') a TCR β variable region sequence, then optionally a TCR β constant domain sequence, a TCR α variable region sequence, then optionally a TCR constant domain sequence, optionally a binder , which is then encoded by a nucleic acid comprising an Fc sequence. In various embodiments, constructs encoding TCR-Fc are preceded by a signal sequence, eg, a secretion signal sequence, to render them selectable.

In another embodiment, the POI is a TCR-Fc, wherein the TCR-Fc comprises a TCR γ chain fused to an Fc sequence and a TCR δ chain variable region fused to an Fc sequence to form a γδ TCR-Fc. includes In certain embodiments, a γδ TCR-Fc comprises two polypeptides: (1) a TCR γ chain variable region fused to a TCR γ chain constant domain fused to an Fc region, and (2) a TCR fused to an Fc sequence. TCR δ chain variable region fused to δ chain constant domain.

T cell receptor variable regions may be identified and/or cloned by any method known in the art. A T cell receptor variable region of a desired protein can be obtained, for example, by expressing rearranged T cell receptor variable region DNA in a cell fused to a human Fc sequence. A rearranged T cell receptor variable region specific for a particular antigen can be obtained by any suitable method known in the art (see references below), for example, by exposing a mouse to the antigen, isolating T cells from the mouse, and It can be obtained by making a hybridoma of a T cell and screening the hybridoma with a desired antigen to obtain a desired hybridoma. A rearranged T cell variable region specific for a desired antigen can be cloned from the desired hybridoma(s). T cell receptor variable regions specific for an antigen can also be identified using phage display technology, eg, as provided in the references below. The variable region can then be cloned and fused to, for example, a human Fc, to create a protein of interest capable of binding a cell surface capture molecule, an FcγR.

Methods for identifying and/or cloning T cell receptor variable regions are described, for example, in U.S. Patent No. 5,635,354 (Primers and Cloning Methods); Genevee et al. (1992) An experimentally validated panel of subfamily-specific oligonucleotide primers (Vα1-w29/Vβ1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction, Eur. J. Immunol. 22:1261-1269 (primers and cloning methods); Gorski et al. (1994) Circulating T cell Repertoire complexity in Normal Individuals and Bone Marrow Recipients Analyzed by CDR3 Size Spectratyping, J. Immunol. 152:5109-5119 (primers and cloning methods); Johnston, S. et al. (1995) A novel method for sequencing members of multi-gene families, Nucleic Acids Res. 23/15:3074-3075 (primers and cloning methods); Pannetier et al. (1995) T-cell repertoire diversity and clonal expansions in normal and clinical samples, Immunology Today 16/4:176-181 (cloning methods); Hinz, T. and Kabelitz, D. (2000) Identification of the T-cell receptor alpha variable (TRAV) gene(s) in T-cell malignancies, J. Immunol. Methods 246: 145-148 (cloning methods); van Dongen et al. (2002) Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: US Pat. No. 6,623,957 (cloning methods and primers); Report of the BIOMED-2 Concerted Action BMH4-CT98-3936, Leukemia 17:2257-2317 (primers and cloning methods); Hodges et al. (2002) Diagnostic role of tests for T cell receptor (TCR) genes, J. Clin. Pathol. 56: 1-11 (cloning method); Moysey, R. et al. (2004) Amplification and one-step expression cloning of human T cell receptor genes, Anal. Biochem. 326:284-286 (cloning methods); Fernandes et al. (2005) Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-cell receptor νβ-Chain Repertoire, Clin. Diag. Lab. Immunol. 12/4:477-483 (primers and cloning methods); Li, Y. et al. (2005) Directed evolution of human T-cell receptors with picomolar affinities by phage display, Nature Biotech. 23/3:349-354 (primers and cloning methods); Wlodarski et al. (2005) Pathologic clonal cytotoxic T-cell responses: nonrandom nature of the T-cell receptor restriction in large granular lymphocyte leukemia, Blood 106/8:2769-2780 (cloning method); Wlodarski et al. (2006) Molecular strategies for detection and quantitation of clonal cytotoxic T-cell responses in aplastic anemia and myelodysplastic syndrome, Blood 108/8:2632-2641 (primers and cloning methods); Boria et al. (2008) Primer sets for cloning the human repertoire of T cell receptor variable regions, BMC Immunology 9:50 (primers and cloning methods); Richman, S. and Kranz, D. (2007) Display, engineering, and applications of antigen-specific T cell receptors, Biomolecular Engineering 24:361-373 (cloning methods). Examples of sTCRs are described in, for example, U.S. Patent Nos. 6,080,840 and 7,329,731; and Laugel, B et al. (2005) Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition, J. Biol. Chem. 280:1882-1892; incorporated herein by reference. Fc sequences are disclosed herein; Examples of Fc sequences, and their use in fusion proteins, are provided, for example, in US Pat. No. 6,927,044 to Stahl et al. All of the above references are incorporated herein by reference.

In a further embodiment of the invention, the cell surface capture molecule is designed to engage and display the desired protein that normally cannot bind to the FcγR capture molecule with sufficient affinity or binds with low affinity. The desired protein includes IgG4 and IgG2 molecules. Thus, modular capture molecules were designed and built based on the ScFv domain fused to the FcγR transmembrane and cytoplasmic domains. The ScFv domain is derived from a high affinity anti-humanFc antibody and contains a heavy chain variable domain fused to a light chain variable domain. The FcγR-TM-cytoplasmic domain was used to allow proper insertion and orientation at the plasma membrane. The ScFv-FcγR-TM-cyto fusion protein binds IgG4 and other Fc containing molecules, as well as IgG2 and IgG1 subtypes, and said heterodimer comprising at least one wild-type CH3 domain (e.g., a bispecific antibody) However, other CH3 domains may contain Fc*-type substitutions.

In a further embodiment of the invention, the cell surface capture molecule is a modified CH3 domain comprising the H95R and Y96F amino acid substitutions (numbering is based on the IMGT system), such as an Fc* polypeptide, e.g. SEQ ID NO : 42 and designed to display it. Such desired proteins include transfer antibodies, such as antibody heterotetramers useful in the preparation of bispecific antibodies, as are commonly described in United States Patent Application Publication No. US 2010/0331527Al on Dec. 30, 2010, which is incorporated herein in its entirety. included by reference. Thus, modular capture molecules were designed and built based on the ScFv* domain fused to the FcγR transmembrane and cytoplasmic domains. The ScFv* domain is derived from a high affinity anti-Fc* antibody and contains a heavy chain variable domain fused to a light chain variable domain. The FcγR-TM-cytoplasmic domain was used to allow proper insertion and orientation at the plasma membrane. The ScFv*-FcγR-TM-cyto fusion protein binds to any Fc*-containing molecule, such as wild-type IgG3, and heterodimers of IgG4, IgG2, and IgG1, which contain at least one Fc* polypeptide sequence.

Example

The following examples are offered to provide those skilled in the art with a complete disclosure and explanation of how to make and use the methods and compositions of this invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1

Structure of pTE084. pTE084 was constructed by ligating the 1,436 bp Xba I fragment of pCAE1OO encoding human FcγRI (hFcγRI; GenBank accession number M21091) to the Xba I site of pRG821. The orientation of hFcγRI in the preferred plasmid resulting from the ligation was examined by restriction mapping to Not I, Pst I, Eco Rl, and Stu I. pTE084 was designed for high expression levels of hFcγRI, a high affinity cell surface receptor for the Fc domain of human IgG. It contains two independent expression cassettes. One cassette is the hFcγRI gene driven by the CMV-MIE promoter and the second cassette is the neomycin phosphotransferase II (npt) gene, which confers resistance to G418 and is driven by the SV40 late promoter.

Structure of a CHO K1 derivative expressing hFcγRI. CHO K1 cells (4 x 10 ⁶ ) were transfected with pTE084 using Lipofectamine™ (Life Technologies; Rockville, MD) according to the manufacturer's suggestion. Cells were cultured in culture medium (10% fetal bovine serum, 90% Ham's F-12, 2 mM L-glutamine; all reagents were from Life Technologies, Rockville, MD) containing 500 μg/ml G418 (Life Technologies) for 15 days. left for days Cells that survived the G418 selection were trypsinized, pooled, and stained with FITC-conjugated human IgG, Fc fragment (FITC-hFc; Jackson ImmunoResearch Laboratories, West Grove, PA). Briefly, cells grown in 10 cm culture plates were washed once with Dulbecco's phosphate-buffered saline (PBS) without calcium chloride and magnesium chloride (Life Technologies). 3 milliliters of 0.25% trypsin (Life Technologies) was added to each plate. The plate was agitated until the cells detached from the plate. Ten milliliters of culture medium was immediately added to each plate of dissociated cells. Cells were then harvested by centrifugation at 1,000 xg for 4 minutes. After removal of the supernatant, cells were resuspended in 4 ml of 2 μg/ml FITC-hFc diluted in culture medium. Cells were then placed on a platform shaker and stained for 1 hour at room temperature. To remove unbound FITC-hFc, cells were washed twice with 20 ml PBS. The extent of FITC-hFc labeling on cells was measured by flow cytometry on a MOFLO™ cell sorting device (Cytomation; Fort Collins, CO). FITC-hFc did not stain the mock-transfected parental CHO K1 cells but gave rise to a distribution of fluorescence in the G418-resistant, pTE084-transfected pool. The most fluorescent cells in the top 1% of the selected pool were placed in 96-well plates at 1 cell/well by cellular assay. After 9 days, 88 cell clones from 96-well plates were expanded to 24-well plates. After 3 days, cells in each well were washed once with 1 ml PBS, stained with 0.5 ml 2 μg/ml FITC-hFc for 1 hour, washed twice with 1 ml PBS and cell surface staining under a fluorescence microscope. inspected. The 33rd most fluorescent clone was selected, expanded and then screened by flow cytometry.

Diffusion of the secreted protein between expressing and non-expressing cells among cells was blocked by adding IgG: hFcγRI As all cells of the clonal cell line express cell surface hFcγRI, they all consist of IgG or the Fc domain of IgG. Possess the ability to bind fusion proteins. Since hFcγRI binds IgG of various species (van de Winkel and Anderson, 1991), binding of a panel of animal IgGs to proteins containing a human IgG1 (hIgG1) Fc tag (4SC622) to hFcγRI-expressing cells was tested for its ability to block 4SC622 is a chimeric molecule composed of an IL-2Rγ extracellular domain fused to an hIL-4Rγ extracellular domain then fused to a hIgG1-Fc domain. In this experiment, cultures of hFcγRI-expressing cell lines selected from CHO K1 cells stably transfected with RGC1, pTE084 were cultured in a 37° C. tissue culture incubator in the presence or absence of 1 mg/ml IgG of different species at 1 μg for 18 hours. /ml incubated with 4SC622.

Cell surface binding of 4SC622 was determined by phycoerythrin-conjugates specific to the hIL-2Rγ component of 4SC622 (BD Pharmingen; San Diego, CA) following the procedure outlined for cell staining of washed cells with FITC-hFc. It was determined by flow cytometry after staining with gated mouse IgG1 monoclonal AG 184 (PE-AG184).

It was found that hIgG completely blocks 4SC622 from binding to hFcγR1 expressed on the surface of RGC1. Rat, rabbit and dog-derived IgG also effectively blocked binding whereas bovine and sheep-derived IgG did not. The ability of the exogenously added rat IgG to block binding of the exogenously added Fc-tagged protein (4SC622) to the cell surface hFcγRI suggests that the rat IgG also affects cells expressing the hIgG1 Fc-tagged protein at different levels. It is suggested that transmission can be blocked between To test this, two cell lines were generated from RGC1 that could be distinguished by the presence or absence of green fluorescent protein (EGFP). Briefly, to mark RGC1 cells with EGFP, 2 x 10 ⁶ RGC1 cells were transfected with 0.5 mg PTE073 encoding hygromycin B phosphotransferase driven by the phosphoglycerate kinase promoter and the CMV-MIE promoter. was co-transfected with 5 mg pRG816-EGFP encoding the EGFP gene driven by Transfected cells were selected with 200 μg/ml Hygromycin B (Sigma; St. Louis, Mo.) for 2 weeks. Green fluorescent cells were isolated by flow cytometry. One EGFP and hFcγRI-expressing clone, RGC2, was used in cell mixing experiments. Another cell line used in these experiments, RGC4, was generated by stable transfection of RGC1 with the plasmid pEE14.1-622. pEE14.1-622 is a plasmid in which the expression of 4SC622 is driven by the CMV-MIE promoter and contains a glutamine synthetase chibi gene, which confers resistance to the analog methionine sulfoximine (MSX) and has stable integration Allow selection of events. RGC4 cells express hFcγRI on the cell surface and secrete the hIgG1 Fc-tagged protein 4SC622. One plate of mixed cells containing 50% RGC2 and 50% RGC4 cells was incubated with 1 mg/ml rat IgG for 18 hours before staining with PE-AG184 and then examined by flow cytometry. EGFP fluorescence of RGC2 cells indicates that RGC2 cells also bind exogenously added 4SC622 (1 μg/ml) as indicated by an increase in PE-AG184 fluorescence. RGC4 did not fluoresce in the EGFP gate. Importantly, exogenously added rat IgG did not reduce the percentage of RGC4 cells that stained positive for cell surface 4SC622, suggesting that binding of 4SC622 to hFcγRI occurs while the protein is translocating to the cell surface. When RGC2 and RGC4 cells were mixed, 4SC622 protein was secreted from RGC4 cells accumulated in the medium and bound to the majority of RGC2 cells. However, addition of 1 mg/ml rat IgG significantly reduced the percentage of RGC2 cells bound to 4SC622, indicating that rat IgG blocks the transfer of secreted hIgG1 Fc-tagged proteins from expressing cells to non-expressing cells. Proven.

Example 2: Cell surface fluorescence correlates with the expression level of 4SC622

RGC1 cells (4 x 10 ⁶ ) were transfected with pEE14.1-622 and pools of stable transfectants were cultured in 10% dialyzed fetal bovine serum, 90% glutamine-free Dulbecco's Modified Eagle's Medium. Eagle's Medium; DMEM), 1 x GS supplement, and 25 μM MSX (all reagents are from JRH Biosciences, Lenexa, KS) for 2 weeks after selection. Rat IgG was added to the culture medium at 1 mg/ml 18 hours prior to immunostaining. Cells were trypsinized, washed with PBS, and polyclonal FITC-conjugated anti-human IgG (H+L) F( ab')2 fragment (Jackson ImmunoResearch Laboratories) was stained with 1.5 μg/ml. Cell staining was then analyzed by flow cytometry. The distribution of fluorescence suggests that the selected pool contains cells with a wide range of 4SC622 expression levels. Cells in the top 3% (R3 bracket), 7-11% (R5 bracket), and 15-19% (R7 bracket) for immunofluorescence were sorted into three separate pools and expanded for 9 days. Average 4SC622 production per cell for the pool was determined by measuring cell numbers and 4SC622 levels in the medium after 3 days of growth by an immuno-based Pandex assay (Idexx; Westbrook, ME) according to the manufacturer's recommendations. In the Pandex assay, 4SC622 was captured from the medium using fluoricon polystyrene assay particles coated with a goat anti-human IgG, g-chain specific antibody (Sigma), and FITC-conjugated goat anti-human IgG , Fc specific (Sigma) was used to detect bead-bound 4SC622. A known amount of purified 4SC622 was included in the calibration assay. It was found that cells in the top 3%, 7-11%, and 15-19% pools produced 4SC622 at 1.42, 0.36, and 0.22 pg/cell/day, respectively. Thus, there was an association between cell surface 4SC622 staining and specific protein production. This result can be obtained by isolating the cells in which individual cells expressing 4SC622 at high levels stained the brightest with a polyclonal FITC-conjugated anti-human IgG (H+L) F(ab')2 fragment. suggest that it may be

Example 3: Isolation of expression clones in the RGC1:IL-4 trap

To directly demonstrate the efficiency of our methodology in generating clonal cell lines that produce high levels of secreted protein, a clone 4SC622 producing cell line was generated from RGC1. RGC1 cells (4 x 10 ⁶ ) were transfected with pEE14.1-622 and selected for 2 weeks with 25 μM MSX to obtain a pool of stable transfectants. MSX-resistant cells were pooled and incubated with 1 mg/ml human IgG for 18 hours before staining with PE-AG184. Six cells of the top 5% gate were isolated and expanded, as determined by flow cytometry analysis of cell surface 4SC622 staining. 4SC622 production was determined from six clonal cell lines and selected colonies were handpicked and then compared to 4SC622 of clones obtained by dilution cloning and amplification. One RGC1-derived clone, RGC4, produced 4SC622 at 12 pg/cell/day. This level is similar to the best 4SC622 producer isolated by hand-picking and analyzing 2,700 clones. Thus, compared to hand-picked colonies, the methodology outlined herein proves to be much more efficient in screening and cloning high producers.

VEGF trap. Plasmids pTE080 and pTE081 encode genes for the VEGF traps, hVEGF-R1R2 and hVEGF-R1R3. hVEGF-R1R2 is a chimeric molecule composed of a first Ig domain of hVEGFRI fused to a second Ig domain of hVEGFR2 fused to a hIg1 Fc domain. hVEGF-R1R3 is a chimeric molecule composed of a first Ig domain of hVEGFI fused to a second Ig domain of hVEGFR3 fused to a hIgG1-Fc domain. In this plasmid, the gene for the VEGF trap is driven by the CMV-MIE promoter and the glutamine synthetase chibi gene, which confers resistance to MSX and is expressed for selection of stable integration events. RGC1 cells were transfected with any of these plasmids and grown in medium containing 25 μM MSX for 2 weeks to select for cells into which the plasmid stably integrates. MSX-resistant cells were incubated with 0.1 μg/ml IgG2a and mouse IgG3 for 18 hours before staining with 1.5 μg/ml polyclonal FITC-conjugated anti-human IgG (H+L) F(ab')2 fragment did Cells were stained for 1 hour then washed twice with PBS before flow cytometry. Single cells were sorted from pools of cells in the most fluorescent 1% into 96-well tissue culture plates. Cells in individual wells were expanded and their productivity was determined by Pandex assay. RGC-derived clones expressing both hVEGF-R1R2 and hVEGF-R1R3 had higher specific productivity and were isolated by screening fewer clones compared to the highest expressing hand-picked MSX-resistant colonies. See Table 1.

Table I
Comparison of specific productivity protein temporary
(μg/ml) Hand-picked CHO K1
stable cell line RGC1-derived
stable cell line Sp. Prod.
(pg/cell/day) screened
clone# Sp. Prod.
(pg/cell/day) screened
clone# 4SC622 1.1 12 2700 12 6 hVEGF-R1R2 33 68 190 77 62 hVEGF-R1R3 27 5 100 22.6 42

Example 4 : cell surface- combined hIgG1 Fc - tagged protein is to RGC1 due to internalized

hFcγRI is known to trigger the internalization of its cell surface-bound ligand. To assay whether RGC1 cells can internalize cell surface-bound 4SC622, 1 μg/ml 4SC622 was added to RGC1 cells for 1 hour, at which time cells were immediately processed for 4SC622 immunostaining with PE-AG184 and flow cytometry. did 93 percent of the cells stained positive for cell surface 4SC622. Alternatively, 1 μg/ml 4SC622 was added to RGC1 cells for 1 hour, at which time the cells were washed and incubated with PE-AG184 in 4SC622-free culture medium for 18 hours. Flow cytometry after immunostaining for 4SC622 showed that 9% of the cells retained 4SC622 on the cell surface. To further characterize the loss of surface-bound 4SC622, purified 4SC622 protein was added to the medium of RGC1 and parental CHO K1 cells, then the level of 4SC622 in the medium was measured over time. 4SC622 added at 2 μg/ml to the culture medium in 10 cm plates was much lower in the RGC1 conditioned medium after 3 days culture compared to the CHO K1 control. These results indicate that the concentration of 4SC622 in the culture medium is reduced by the presence of hFcγRI on the cell surface. The results suggest that the depletion of 4SC622 in the medium is a result of hFcγRI-4SC622 complex internalization. Internalization of this receptor-ligand complex may facilitate effective removal of all 4SC622 from non-expressing cells in the presence of blocking IgG during the 18-hour blocking step.

Example 5: Rescue of CHO K1 cell line with inducible hFcγRI expression

A flow cytometry-based autologous secretion trap using hFcγRI allows rapid isolation of high expressing clones. However, when hFcγRI mediates the turnover of an Fc-tagged protein, then the perceived product of the secreted protein by the expressed hFcγRI expressing cells is higher if hFcγRI can be restricted during production. At this end, a CHO K1 cell line was constructed in which expression of hFcγRI was induced by tetracycline, or the analogue doxycycline. In this system, CHO K1 cells were first engineered to express tetracycline repressor protein (TetR) and hFcγRI was placed under the transcriptional control of a promoter whose activity is regulated by TetR. Two tandem TetR operators (TetO) were placed immediately downstream of the CMV-MIE promoter/enhancer in pTE084 to generate pTE158. Transcription of hFcγRI from the CMV-MIE promoter in pTE158 was blocked by TetR in the absence of tetracycline or any other suitable inducer. In the presence of the inducer, the TetR protein was unable to bind TetO and transcription of hFcγRI occurred.

CHO K1 cells were transfected with pcDNA6/TR and a plasmid conferring resistance to blasticidin in which expression of TetR originates from the CMV-MIE promoter (Invitrogen; Carlsbad, Calif.). After 2 weeks of selection with 2.5 μg/ml blasticidin (Invitrogen), stable transfectants were pooled. This pool was then transfected with pTE158, a plasmid conferring resistance to G418 in which expression of hFcγRI is dependent on the CMV-MIE/TetO hybrid promoter. Cells serially transfected with pcDNA6/TR and pTE158 were selected with 400 μg/ml G418 and 2.5 μg/ml blasticidin for 12 days and then pooled. Pools were evoked for 2 days by the addition of 1 μg/ml doxycycline, then stained with FITC-hFc to identify cells expressing hFcγRI. The top 5% of cells expressing hFcγRI were harvested as pools, expanded for 6 days without doxycycline, and restained with FITC-hFc for the presence of hFcγRI. Cells not stained for hFcγRI were harvested and expanded for 3 days in culture medium containing 1 μg/ml doxycycline. Pools were then stained for the presence of hFcγRI and separated by flow cytometry. Cells expressing the highest level of hFcγRI (top 1%) were sorted into 96 well plates at one cell per well. These cells probably contain cells with low non-induced expression levels of FcγR1 and high induced levels of FcγR1. After expansion, induction of hFcγRI by doxycycline in 20 clones was confirmed by immunostaining with FITC-hFc and flow cytometry. One clone was selected for additional properties and named RGC10.

In the absence of doxycycline, RGC10 did not express detectable levels of hFcγRI, whereas high levels of hFcγRI were observed for 3 days in cells induced with 1 μg/ml doxycycline. The average fluorescence of RGC10 cells increased more than 1,000-fold after challenge with doxycycline.

Example 6: Isolation of 4SC622-producing cell lines of RGC10

RGC10 cells were transfected with pEE14.1-622, and MSX-resistant cells were selected with 25 mM MSX for 2 weeks and then pooled. Expression of hFcγRI was induced by the addition of 1 μg/ml doxycycline to the culture medium for 3 days. 1 mg/ml rat IgG was stained with polyclonal FITC-conjugated anti-human IgG (H+L) F(ab')2 fragment containing doxycycline 18 hours before analysis by flow cytometry. added to the culture medium. Cells expressing the highest level of 4SC622 (top 1%) were sorted into 96 well plates at 1 cell per well. Staining with polyclonal FITC-conjugated anti-human IgG (H+L) F(ab')2 fragment failed to detect cell surface bound 4SC622 without eliciting hFcγRI expression by doxycycline do. 60 clones were expanded in the absence of doxycycline. The specific productivity of the 13 highest producers was determined by Pandex assay. The specific productivity of clone 1C2 is 17.8 pg/cell/day, much better than the 12 pg/cell/day observed for the best 4SC622 cell line previously isolated using the unregulated hFcγRI cell line RGC1.

Example 7: Sp2 /0 myeloma cells are cell surface capture Can be engineered to express proteins

In this example, the Sp2/0-Ag14 myeloma cell line was engineered to stably express hFcγRI to demonstrate that the autocrine trap method is applicable to cell lines other than CHO. The gene for hFcγRI was introduced into myeloma cells by retrovirus infection. Plasmid pLXRN (Clontech; Palo Alto, CA), encoding the hFcγRI gene using a retroviral DNA vector in which the desired gene may be expressed from the upstream Moloney murine sarcoma virus long terminal repeat (MoMuSV LTR) promoter A retrovirus was created. A 1,363 bp XhoI fragment of pTE084, encoding the human FcγRI gene, was cloned into the XhoI site of pLXRN. A plasmid whose hFcγRI cDNA expression was dependent on the MoMuSV LTR was selected and named pTE255.

A pantropic retrovirus for expression of hFcγRI was generated essentially following the manufacturer's guidelines. Packaging cell line GP-293, a HEK 293-based cell line stably expressing viral gag and pol proteins (Clontech; Palo Alto, Calif.), was co-transfected with 10 mg of pVSV-G and pTE255, respectively. The plasmid pVSV-G allows expression of the viral envelope protein VSV-G, which confers a broad host range to infectious particles.

Structure of Sp2 - hFcγRI -4. 1×10 ⁷ Sp2/0-Ag14 myeloma cells (American Type Culture Collection; Manassas, Va.) were infected with a panaffinity hFcγRI retrovirus at a range of approximately 10 infectious particles per cell. On day 3 post infection, cells were stained for 1 hour and then washed twice with PBS before analysis by flow cytometry. The cells expressing hFcγRI, as indicated for bound FITC-hFc, were harvested as pools by flow cytometry. Pools were expanded for 13 days at which time they were stained again with FITC-hFc and cells expressing hFcγRI were harvested as pools by flow cytometry. These sorted cells were cultured in 90% Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum containing 4.5 g/l glucose and 4 mM glutamine for 3 weeks, stained with FITC-hFc, and the top 1% of the population. Cells with average fluorescence in were cloned by single cell sorting. After expansion, 24 clones were examined by flow cytometry for expression of hFcγRI, as described above, and one clone, Sp2-hFcγRI-4, was selected for further characterization.

Isolation of Sp2 - hFcγRI -4 cells expressing the 4SC622 protein. Sp2-hFcγRI-4 cells (1×10 ⁷ ) were transfected with pTE209, a plasmid allowing constitutive expression of 4SC622 from the CMV-MIE promoter and conferring resistance to hygromycin. Transfected cells were placed in medium containing 10% FCS, 90% D-MEM and 400 μg/ml hygromycin for 14 days. Hygromycin-resistant cells were incubated with 1 mg/ml rabbit IgG for 18 hours before staining with polyclonal FITC-conjugated anti-human IgG (H+L) F (ab')2 fragment. Cells were stained for 1 hour prior to analysis by flow cytometry, then washed twice with PBS. Labeled cells were harvested as pools by flow cytometry as described above and then cultured for 5 days and sorted. Cells from the expanded pool, the top 1% population, most of which bound to the polyclonal FITC-conjugated anti-human IgG (H+L) F (ab') 2 fragment, were then cloned by single cell sorting. Production of 4SC622 from 10 clones was analyzed by ELISA and all 10 clones were found to express 4SC622; Clone 5H11 produced 4SC622 at 0.5 pg per cell per day. These data effectively isolated clones secreting 4SC622 by an autocrine trap method from a foreign pool of cells derived from stable transfection of Sp2-hFcγRI-4 cells with pTE209.

To confirm that 4SC622 self-displayed on the surface of myeloma cells expressing both 4SC622 and hFcγRI, clone 5H11 was incubated with 1 mg/ml rabbit IgG for 18 hours and then FITC-conjugated anti-human IgG ( H+L) F(ab')2 fragment was stained and found to display cell surface 4SC622. Displayed under conditions where cross-feeding of the secreted protein is blocked by rabbit IgG, demonstrating the autologous display of 4SC622. These data indicate that the autocrine trap method described above is not limited to CHO cells and may extend to myeloma as well as other cell types.

Example 8. Protein G chimera proteins on the cell surface capture as a protein to function can

To demonstrate the application of the autocrine trap method to cell surface capture proteins other than hFcγRI, a cell line expressing protein G was constructed. Protein G of Streptococcus strain G148 binds all human and mouse IgG subclasses and as such has utility for the isolation of recombinant cells expressing antibodies or IgG Fc fusion proteins. To demonstrate that the Protein G IgG Fc-binding domain can be used as a cell surface capture protein capable of binding all human and mouse IgG subclasses, the inventors fused the Fc-binding domain of Protein G to the hFcγRI transmembrane and intracellular domains. A CHO cell line expressing a chimeric protein composed of was constructed. The Fc binding domain of protein G contains three 55 amino acid long homologous repeat regions (Guss et al., (1986) EMBO 5:1567 and Sjobring et al., (1991) J. Biol. Chem. 266: 399) Each repeat region can bind one IgG Fc. To improve the expression of this chimeric protein in CHO cells, the inventors created a synthetic DNA in which the signal sequence of the mouse ROR1 gene is fused to the Fc binding domain, amino acids 303 to 497 of protein G (accession number X06173) (SEQ ID NO: 1). was composed. This synthetic DNA was generated by a combination of oligonucleotide annealing, gap filling, and PCR amplification. The synthetic DNA was then fused by PCR to DNA encoding the transmembrane and intracellular domains, amino acids 279 to 374 of hFcγRI (SEQ ID NO:2) (Accession No. M21091). The resulting DNA encoding the protein G/hFcγRI chimeric protein was cloned into pTE158 downstream of the CMV-MIE promoter replacing the gene encoding hFcγRI to obtain plasmid pTE300, replacing the gene encoding hFcγRI.

RGC14, a CHO K1 cell line adapted to grow in serum-free medium, was transfected with pTE300 and after 3 days 400 μg/ml G418 was added to the culture medium to select for stable integration of pTE300. Two weeks after the start of selection, cells were stained with FITC-hFc to identify cells expressing hFcγRI. These cells were analyzed by flow cytometry and cells expressing hFcγRI were harvested as pools. Cells were expanded for 10 days and populations of cells expressing hFcγRI were again isolated by flow cytometry. Cells were re-expanded, stained with FITC-hFc, and single cells expressing high levels of the protein G/hFcγRI chimeric protein were isolated by flow cytometry. Single cells that stained positive for FITC-hFc binding were sorted into medium consisting of 10% fetal bovine serum, 90% Ham's F12, and 400 μg/ml G418. After 2 weeks of culture, 48 clones were tested for binding to bovine IgG present in the culture medium by staining with FITC-conjugated anti-bovine IgG F(ab')2 fragment (Jackson ImmunoResearch Laboratories, West Grove, PA). . One clone, RGC18, which stains positively with this antibody, was selected for further characterization.

Isolation of expressing clones in RGC18: RGC18 cells (6×10 ⁶ ) were transfected with pTE209 and selected for integration of the plasmid by growth in 400 μg/ml hygromycin for 18 days. Hygromycin-resistant cells were incubated with 1 mg/ml rabbit IgG for 18 hours before staining with polyclonal FITC-conjugated anti-human IgG (H+L) F (ab')2 fragment. Cells were stained for 1 hour prior to analysis by flow cytometry, then washed twice with PBS. The most fluorescent cells (top 5%) were isolated by single cell sorting and expanded for 3 weeks. Ten clones were tested for 4SC622 secretion. All clones tested secreted high levels of 4SC622, with the best clone, RGC19, having a specific productivity of 6.4 pg/cell/day. This result demonstrated that 4SC622-expressing cells were effectively separated from the foreign pool of cells derived from stable transfection of RGC18 with pTE209 by the autocrine trap method. Moreover, these data clearly demonstrated that fragments of protein G are designed to contain a signal sequence and a transmembrane domain and function as cell surface capture proteins.

To confirm that 4SC622 is self-displayed on the surface of RGC19 cells expressing both the protein G/hFcγRI chimeric protein and 4SC622, RGC19 was incubated with 1 mg/ml rabbit IgG for 18 hours and then FITC-conjugated antibody - Stained with human IgG (H+L) F(ab')2 fragment and analyzed by flow cytometry. RGC19 cells were found to possess cell surface 4SC622 under these conditions where cross-uptake was blocked by rabbit IgG, suggesting autologous display of 4SC622. Rabbit IgG effectively blocked the binding of exogenous 4SC622 protein to RGC18 cells, but did not block the display of 4SC622 on the cell surface of cells expressing 4SC622. These data demonstrated that the properties of the protein G/hFcγRI chimeric protein were similar to hFcγRI as cell surface capture proteins, suggesting that the autocrine trap method could use other proteins as cell surface capture proteins.

Example 9: Isolation of antibody-producing cells from RGC10

To demonstrate the utility of the autocrine trap method for the isolation of CHO cell lines expressing recombinant antibodies, the inventors cloned DNA encoding the KD5 hybridoma variable light chain and variable heavy chain genes. KD5 is a hybridoma expressing a monoclonal antibody specific for the human Tie-2 receptor.

Mouse IgG constant domain gene sequences were cloned from 500ng mouse spleen polyA+ RNA (Clontech, Palo Alto, Calif.). Single-stranded cDNA was synthesized using the Superscript First-Strand Synthesis System for RT-PCR and primed with 50 ng random hexamers (Invitrogen Life Technologies, Carlsbad, CA). The mouse kappa light chain constant DNA sequence (Accession No. Z37499) was combined with primers 5′ mCLK1 (Z37499) (5′-CGGGCTGATG CTGCACCAAC TGTATCCATC TTC-3′) (SEQ ID NO:3) and 3′ mCLK1 (Z37499) (5′-ACACTCTCCC CTGTTGAAGC TCTTGACAAT GGG-3') (SEQ ID NO:4) was amplified from this cDNA by PCR. The mouse IgG2 constant domain DNA sequence (accession number AJ294738) was also subjected to primers 5′ mCH2a (AJ294738) (5′-GCCAAAACAA CAGCCCCATC GGTCTATCCA C-3′) (SEQ ID NO:5) and 3′ mCH2a (AJ294738) (5′- TCATTTACCC GGAGTCCGGG AGAAGCTCTT AGTCG-3') (SEQ ID NO:6) was amplified from this cDNA by PCR. The PCR product was cloned into pCR2.1-TOPO using the TOPO TA Cloning kit (Invitrogen Life Technologies, Carlsbad, Calif.) and the sequence of the constant domain was confirmed.

The KD5 variable region gene was amplified by RT-PCR from KD5 hybridoma mRNA and cloned into pCR2.1-TOPO using a mixture of heavy and light chain variable region primers from Amersham-Pharmacia Biotech (Piscataway, NJ). The variable heavy chain gene was sequenced with primers 5' BspMI/KD5VH N-terminus (5'-GAGAGTACCT GCGTCATGCA GATGTGAAAC TGCAGGAGTC TGGCCCT-3') (SEQ ID NO:7) and 3' BspM I/KD5VH C-terminus (5'-GAGAGACCTG CGTCAGCTGA GGAGACGGTG ACCGTGGT-3') (SEQ ID NO:8) was PCR amplified using the pCR2.1-TOPO cloned variable region as template, digested with BspMI and primer 5' BsaI/CH2a N-terminus (5'-GAGAGGGTCT CACAGCCAAA ACAACAGCCC CATCG-3') (SEQ ID NO:9) and 3' BsaI/ CH2a C-terminal (5'-GAGAGGGTCT CCGGCCGCTC ATTTACCCGG AGTCCGGG AGAA-3') (SEQ ID NO: 10) ligated to the PCR fragment of the IgG2a constant heavy chain gene. This fragment was then ligated into the BspMI and NotI sites of pRG882. The resulting plasmid, pTE317, was capable of expressing the KD5 recombinant heavy chain gene from the CMV-MIE promoter fused to the mROR1 signal sequence. The variable light chain gene was sequenced with primers 5' BsmBI/KD5VL N-terminus (5'-GAGAGCGTCT CATGCAGACA TCCAGATGAC CCAGTCTCCA-3') (SEQ ID NO:11) and 3' BsmBI/KD5VL C-terminus (5'-GAGAGCGTCT CACAGCCCGT TTTATTTCCA GCTTGGTCCC- 3') (SEQ ID NO: 12) was PCR amplified using the pCR2.1-TOPO cloned variable region as template, digested with BsmBI and primers 5' BsaI/CLK N-terminus (5'-GAGAGGGTCT CAGCTGATGC TGCACCAACT GTATCC-3') (SEQ ID NO:13) and BsaI-cleaved kappa constant amplified with 3' BsaI/CLK C-terminus (5'-GAGAGGGTCT CAGGCCGCTC AACACTCTCC CCTGTTGAAG CTCTTGAC-3') (SEQ ID NO:14) The light chain gene PCR fragment was ligated. This fragment was then ligated into the BspMI and NotI sites of pRG882. The resulting plasmid, pTE316, is capable of expressing the KD5 recombinant light chain gene from the CMV-MIE promoter, fused to the mROR1 signal sequence.

The 1450 bp EcoRI-NotI fragment of pTE317, encoding the KD5 heavy chain gene, was cloned into pRG980, the EcoRI and NotI sites of the vector conferring resistance to hygromycin and expression of the recombinant gene against the UbC promoter yielded plasmid pTE322 did Similarly, the 750 bp EcoRI-NotI fragment of pTE316, encoding the KD5 light chain gene, was transformed into pRG985, the EcoRI and NotI sites of the vector conferring resistance to puromycin and expression of the recombinant gene against the UbC promoter to yield plasmid pTE324. cloned into. RGC10 cells (5 x 10 ⁶ ) were transfected with 3 μg pTE322 and 3 μg pTE322 and grown for 14 days in F12 medium supplemented with 10% fetal bovine serum with 20 μg puromycin and 400 μg/ml hygromycin. was selected for integration of the plasmid by. Expression of hFcγRI was induced by addition of 1 μg/ml doxycycline to the culture medium for 3 days. Dual-resistant cells were stained with goat polyclonal FITC-conjugated anti-mouse IgG (Fey) F (ab') 2 fragment (Jackson ImmunoResearch Laboratories, West Grove, PA) at 1 mg/ml rabbit IgG for 18 hours before staining. incubated with. Cells were stained for 1 hour prior to analysis by flow cytometry, then washed twice with PBS. The most fluorescent cells (top 5%) were isolated as a pool and expanded for 10 days, after which the protocol was repeated but the top 1% most fluorescent cells were isolated as a pool. This pool was expanded for 10 days at which time the top 0.1% most fluorescent cells were isolated as single cells in 96-well plates. Clones were analyzed by ELISA for expression of antibody and 7 clones were selected from the 53 clones analyzed. The average specific productivity of these clones was 35 pg/cell/day and the best clone expressed recombinant KD5 monoclonal antibody at 54 pg/cell/day.

Example 10 : FASTR ™ is CSCP not affected by expression level screen

To demonstrate that the level of expression of CSCP does not significantly affect the ability to isolate cells expressing the relevant sPOI, FASTR™ was tested for the same expression in two different host cell lines expressing the same CSCP but compared at high or low levels, respectively. Screen for sPOI.

The FASTR™ host cell line RGC10 was selected for high level expression of the hFcγRI protein by stable integration of pTE158 and found to contain 40 hFcγRI integrated gene colonies. A new cell line expressing the hFcγRI protein at low levels, RS527, was generated from CHO K1 after stable transfection and selection for single copy gene integration. RS527 cells expressed significantly less hFcγRI protein than RGC10 cells as determined by Western blot analysis of whole cell lysates of the FASTR™ cell line.

Briefly, RGC10 and RS527 cells were transfected with pTE462, a plasmid capable of expressing the secreted hFc-fusion protein Rc1-hFc and conferring resistance to hygromycin. Transfected cultures were selected with hygromycin for 2 weeks. Hygromycin-resistant cells were induced with 1 μg/ml doxycycline (Dox) and blocked overnight with rabbit IgG, according to the FASTR™ method described herein. The next day RGC10/pTE462 and RS527/pTE462 cultures were stained with a FITC-conjugated antibody specific for hFc and then analyzed by flow cytometry. Three cell bins R4, R5, and R6, representing cells with low, medium, and high fluorescence, respectively, were sorted from each host cell line and expanded in tissue culture.

To compare Rc1-hFc protein production levels with six cell bins, six cultures were set up using the same number of cells for each bin. After 3 days, the conditioned medium was harvested. Rc1-hFc protein titers in conditioned media were determined by ELISA and plotted against the mean fluorescence of each cell bin. For both the RGC10 and RS527 host cell lines, there was a similar correlation between mean fluorescence (amount of Rc1-hFc displayed on the cell surface) and sPOI protein production levels of the isolated cell pools. More importantly, sPOI titers were similar in the two highly fluorescent R6 bins derived from RGC10 and RS527. This data demonstrates that the expression level of CSCP in the FASTR™ host cell line did not significantly affect the use of this host to isolate transfected cells based on the expression level of sPOI.

Example 11: Tie2 receptor as a cell surface capture protein

Cell surface capture proteins (CSCPs) other than FcγR1 can be used in the methods described herein. In this example, the Tie2 receptor functions as a CSCP and is used to isolate cells expressing a Tie-specific ScFv _C1b -Fc fusion protein made from a C1b monoclonal antibody that specifically binds to the extracellular domain of the Tie2 receptor. . Although the CSCP for ScFv _C1b -Fc can be hFcgRI, this example demonstrates that Tie2 can be used as the CSCP for ScFv _C1b -Fc.

To construct the inducible Tie2 CSCP cell line, CHO K1 was first stably transfected with the TetR plasmid pcDNA6/TR. The blasticidin-resistant cell pool was then stably transfected with pTE259, a plasmid allowing inducible expression of a protein consisting of the extracellular and transmembrane domains of Tie2. Induced cell clones were isolated by flow cytometry after staining with an antibody specific for Tie2. The RGC54 clone was selected to study the feasibility of FASTR™ on the expression of ScFv _C1b -Fc.

RGC54 cells were stably transfected with a plasmid capable of expressing the secreted hFc-fusion protein ScFv _C1b -Fc and conferring resistance to hygromycin. Transfected cultures were selected with hygromycin for 2 weeks. Hygromycin-resistant cells were induced with Dox and blocked with 1 mg/ml of purified C1b mAb. C1b monoclonal antibody was the source of the variable region in ScFv _C1b -Fc. The next day, cell pools were stained with a FITC-conjugated antibody specific for hFc and then analyzed by flow cytometry. Three cell bins, R6, R7, and R8, representing cells with high, medium, and low fluorescence, respectively, were sorted and expanded in tissue culture. Three cultures were set up using the same number of cells for each bin to determine ScFv _Clb -Fc protein production as determined by ELISA. There was a correlation between the mean fluorescence of the isolated cell pool (the amount of ScFv _C1b -Fc binding to Tie2 on the cell surface) and the level of ScFvC1b-Fc protein production.

These data indicate that CSCPs other than hFcγRI can serve as CSCPs, and also suggest that any receptor can be converted to CSCPs by removal of its cytoplasmic domain. This data also demonstrates that the antigen can be made into CSCP and used in FASTR™ to screen for cells expressing antigen-specific antibody-related molecules.

Example 12: effective FASTR ™ is low affinity having CSCP:sPOI in pairs screen

Angiopoietin-1 is a ligand for the Tie2 receptor. A chimeric protein comprising an angiopoietin-1 receptor binding domain and hFc (FD1-hFc) binds Tie2 with an affinity constant of 174 nM as determined by BIAcore™. FD1-hFc and Tie2 were selected as sPOI and CSCP, respectively, to determine if a minimum affinity between CSCP and sPOI is required for FASTR™ screening.

In cell application experiments, exogenously added FD1-hFc specifically bound to RGC54 cells via Tie2. To determine if the affinity between Tie2 and FD1-hFc is sufficient to permit FASTR™ screening, RGC54 cells were transfected with pTE942, which expresses the secreted hFc-fusion protein FD1-hFc and can confer resistance to hygromycin. Stably transfected with the plasmid. Transfected cultures were selected with hygromycin for 2 weeks. Hygromycin-resistant cells were induced with Dox and blocked with 1 mg/ml of purified FD1-mFc containing mouse IgG1 Fc. The next day, cell pools were stained with a FITC-conjugated antibody specific for hFc and then analyzed by flow cytometry. Three cell bins R6, R7, and R8 displaying high, medium, and low fluorescence, respectively, were harvested. Cultures were set up using the same number of cells for each bin to determine the level of FD1-hFc protein production in the conditioned media as determined by ELISA. There was a correlation between the mean fluorescence of the isolated cell pool (FD1-Fc binding to cell surface-bound Tie2) and the level of FD1-hFc protein production. The bin with the highest fluorescence produced the most FD1-hFc.

This data demonstrates that a low affinity (174 nM KD) CSCP:sPOI pair can be used for effective FASTR™ screening. Importantly, the dissociation t _1/2 for the FD1-Fc:Tie2 binding is less than 2 minutes, suggesting that any _CSCP :sPOI pairing with measurable affinity could work in FASTR™ screening. Moreover, this experiment also indicates that non-FcγRI receptors can also be used as CSCPs to isolate cells expressing the ligand.

Experiment 13: Fusion of transmembrane domain to ScFv creates a functional CSCP

CSCP can be any cell surface-bound protein with measurable affinity for sPOI. To demonstrate this, an entirely synthetic CSCP was constructed by fusing the transmembrane domain of the PDGF receptor to a ScFv containing the variable region of the murine kappa chain-specific monoclonal antibody HB58. A FASTR™ host was constructed to express this chimeric protein (ScFv _HB58 -TM _PDGFR ) and used to isolate cells expressing the angiopoietin-2 FD domain-specific P12 antibody.

The RS655 cell line derived from CHO K1 constitutively expresses the ScFv _HB58 -TM _PDGFR . Cells expressing the ScFv _HB58 -TM _PDGFR can be stained by sequential incubation with the P12 mAb, FD2-hFc, and FITC-conjugated anti-hlgG-P12 captured on the cell surface by the HB58 ScFv to FD2. , which in turn was detected by recognition of the hFc tag. RS656 cells were derived from RS655 cells after stable transfection with a plasmid encoding the gene for eYFP. Nearly 100% of RS656 cells were eYFP-positive and most (76%) retained expression of ScFv _HB58 -TM _PDGFR as detected by binding to FD2-hFc.

RS655 cells were stably transfected with pTE693, a plasmid capable of expressing the heavy and light chains of the P12 antibody and conferring resistance to puromycin. Transfected cultures were selected with puromycin for 2 weeks to obtain a pool of cells (RS655/pTE693) that were foreign with respect to P12 mAb expression.

To determine if the ScFv _HB58 -TM _PDGFR could function as a CSCP and facilitate the separation of antibody-producing cells from non-producers, equal numbers of RS656 cells and RS655/pTE693 cells were mixed and co-cultured. When P12 expressed from RS655/pTE693 cells was allowed to spread and bind to ScFv _HB58 on the surface of RS656 cells, many amber cells were also positive for binding to FD2-hFc. However, ScFv _HB58 on the surface of RS656 bound excessive murine IgG, then only non-yellow cells were positive for binding to FD2-hFc, demonstrating that expressing cells were effectively separated from non-expressing cells.

These data demonstrate that ScFvs can be made from functional CSCPs by targeting them to the cell membrane. The data also indicate that FASTR™ allows cells expressing the secreted antibody to be detected as the antibody's antigen.

Example 14: Desired protein comprising a T cell receptor variable region

A flow cytometry-based autocrine trap (FASTR™) method for isolating high-expressing clones of a cell line expressing a desired protein, TCR-Fc, is prepared in a manner similar to the preparation of cell lines expressing the desired antibody. High expressing clones were identified by screening for cells displaying on their surface the desired TCR-Fc bound to hFcγR.

In this example, the CHO K1 cell line RGC10, which contains inducible FcγR1 as a cell surface capture molecule, is used. RGC10 is made to express recombinant TCR-Fc by cloning the TCR variable region in frame, into a human Fc region, or directly between the TCR variable region and a human Fc region, either in frame or as a joiner sequence.

To create the desired protein, which is a dimer comprising an Fc-linked TCR α variable domain and an Fc-linked TCR β variable domain, RGC10 consists of two vectors, a human Fc sequence capable of expressing a TCR α variable domain fusion protein. transfected with a first vector and a second vector capable of expressing a TCR β domain fusion protein with the same human Fc sequence. Each vector contains a leader sequence 5' to the TCR variable region (eg, a secretion signal sequence), and a selectable marker that is a drug resistance gene. After each vector transfection, cells containing the vector were selected by appropriate drug selection. Selection results in an RGC10 cell line with both the first and second vectors. Cells expressing the desired protein can be detected by one or more of antibodies to the β variable domain, antibodies to the variable domain, and antibodies to the Fc domain.

To make a protein of interest that is a dimer comprising both α and β TCR variable domains fused to Fc, RGC10 is transfected with a single vector encoding the protein of interest consisting of: a leader sequence (e.g., a secretion signal sequence), followed by a TCR variable β domain fused to a linker, wherein the linker is, in turn, fused to a TCR variable domain, which in turn is fused to an Fc sequence. Alternatively, a single vector can be constructed as follows: a leader sequence (e.g., a secretion signal sequence) followed by a TCR variable α domain fused to a linker, which linker, in turn, is fused to a TCR variable β domain. , which in turn is fused to the Fc sequence. Cells expressing the desired protein can be detected by one or more of antibodies to the β variable domain, antibodies to the α variable domain, and antibodies to the Fc domain.

As above, to make a desired protein that also includes a TCR α and/or TCR β constant domain, a TCR variable domain (α or β) is fused to a TCR constant domain (e.g., a TCR variable domain α is a TCR constant domain). domain α, the TCR variable domain β is fused to the TCR constant domain β), the TCR variable + constant domain is fused to the Fc domain either directly or via a linker. Cells expressing the desired protein can be detected by one or more of antibodies to the β variable domain, antibodies to the α variable domain, and antibodies to the Fc domain.

Cells expressing the desired amount of TCR-Fc are prepared by injecting at least one of an antibody against the α variable domain, an antibody against the β variable domain, an antibody against the α constant domain, an antibody against the β constant domain, and an antibody against the Fc domain. isolate using the same procedure used to isolate the 4SC622-producing cell line described herein. Cells expressing the highest level of TCR-Fc are selected as TCR-Fc-producing cell lines.

Example 15: many IgG isotype and for the separation of bispecific antibodies ScFvs -base CSCP

Genetically modified mice in which the immunoglobulin heavy chain VDJ region and the immunoglobulin kappa chain VJ region of the genome are replaced with human orthologs (i.e., the Velocimmune® mouse; U.S. Patent No. 7,105,348, which is incorporated herein by reference in its entirety). ) of human IgG4 protein (hFc, or simply Fc; SEQ ID NO: 26), or human ΔAdpFc polypeptide containing dipeptide mutations (H95R, Y96F by IMGT; also known as Fc*; SEQ ID NO: 42). Monoclonal antibodies were obtained from mice and screened for their ability to bind Fc, Fc*, or antibodies comprising Fc and/or Fc*. Three antibodies capable of binding Fc (Ab1, Ab2, Ab3) and three antibodies capable of binding Fc* (Ab4, Ab5, Ab6) were tested for their ability to bind molecules having one of the following formats: : Fc/Fc, Fc/Fc* (which may be a bispecific antibody), and Fc*/Fc*.

Measurements to determine binding affinity and kinetic constants were made on a Biacore 2000 instrument. Antibodies (Ab1-Ab8, respectively) were captured onto an anti-mouse-Fc sensor surface (Mab capture format), human Fc (SEQ ID NO:26) homodimer, human Fc* homodimer (SEQ ID NO:42), or Fc/Fc* heterodimers were injected onto the surface. Kinetic association (k _a ) and dissociation (k _d ) rate constants were determined by processing the data and fitting them to a 1:1 association model using Scrubber 2.0 curve fitting software. Association dissociation equilibrium constants (K _D ) and dissociation half-lives (t1/2) were calculated from the kinetic rate constants: K _D (M) = k _d / k _a ; and t1/2 (min) = (In2/(60*k _d ). As shown in Table 2, antibodies fall into three distinct categories: Fc specific, Fc* specific, and between Fc and Fc*. Those that did not show discrimination (non-specific) Fc specific antibodies were dependent on the amino acids His 95 and/or Tyr 96 as these antibodies do not bind human Fc* with dipeptide mutations (H95R, Y96F) (H95R, Y96F) Conversely, Fc* specific antibodies are dependent on Arg 95 and/or Phe 96 as these antibodies do not bind wild-type human Fc.

Example 16: Ab2 and Ab2 - originated ScFvs - FcγR Cell lines producing fusion proteins

The heavy and light chains of Fc-specific Ab2 were sequenced. To prepare the recombinant Ab2 antibody, an expression vector plasmid encoding the heavy chain was constructed, and an expression vector plasmid encoding the light chain was constructed. Both vectors allow expression and secretion of each subunit in CHO cells. To express the antibody, both plasmids were transfected into CHO-K1 cells and stable transformants were isolated. Expression of the antibody chains was driven by a constitutive CMV promoter.

Affinity of Antibodies - Surface Plasmon Resonance Studies antibody POI - target ka (M ^-1 s ^-1 ) k d (s ^-1 ) K _D (M) t½ (min) specificity Ab1 Fc/Fc 1.07E+05 3.79E-04 3.54E-09 30 Fc Fc/Fc* 8.16E+04 3.01E-04 3.69E-09 38 Fc*/Fc* NB NB NB NB Ab2 Fc/Fc 7.86E+04 3.50E-05 4.45E-10 330 Fc Fc/Fc* 5.45E+04 1.00-06 1.84E-11 11550 Fc*/Fc* NB NB NB NB Ab3 Fc/Fc 1.77E+05 4.08E-02 2.30E-07 0.3 Fc Fc/Fc* 4.51E+04 2.60E-02 5.77E-07 0.4 Fc*/Fc* NB NB NB NB Ab4 Fc/Fc NB NB NB NB Fc* Fc/Fc* 6.00E+03 1.00E-06 2.00E-10 11550 Fc*/Fc* 2.22E+04 9.56E-06 4.50E-10 1209 Ab5 Fc/Fc NB NB NB NB Fc* Fc/Fc* 3.11E+05 1.00E-06 3.21E-12 11550 Fc*/Fc* 5.57E+05 1.00E-06 1.79E-12 11550 Ab6 Fc/Fc NB NB NB NB Fc* Fc/Fc* 4.48E+05 7.43E-04 1.66E-09 16 Fc*/Fc* 8.73E+05 5.93E-04 6.79E-10 19 Ab7 Fc/Fc 6.02E+05 2.42E-04 4.02E-10 48 non-specific Fc/Fc* 4.90E+05 2.15E-04 4.39E-10 54 Fc*/Fc* 4.46E+05 3.20E-02 7.18E-08 0.4 Ab8 Fc/Fc 2.59E+05 4.88E-04 1.88E-09 24 non-specific Fc/Fc* 1.88E+05 4.02E-04 2.14E-09 29 Fc*/Fc* 4.10E+04 3.90E-02 9.60E-07 0.3

Heavy and light chain sequences were used to develop anti-Fc ScFv surface capture molecules. To prepare nucleic acids encoding Ab2-driven anti-Fc ScFv-FcγR surface capture molecules, Ab2 immunoglobulin heavy chain variable domain (SEQ ID NO:15) and Ab2 immunoglobulin light chain variable domain (SEQ ID NO:16) amino acids The sequence was reverse translated and codon optimized for CHO cell expression. Similarly, the C-terminal portion of human FcγRI was codon optimized for CHO cell expression. The codon-optimized nucleotide sequence was amplified via polymerase chain reaction and ligated to form a contiguous nucleic acid sequence encoding the ScFv-FcγR fusion protein of SEQ ID NO:19 (SEQ ID NO:20).

A nucleic acid encoding the ScFv-FcγR-TM-cyto fusion protein was inserted into an expression vector using standard pcr and restriction endonuclease cloning techniques. The resulting circular plasmid, exemplified by SEQ ID NO:23, contains a beta-lactamase-encoding nucleic acid sequence, and two operons. The first operon contains a nucleic acid sequence encoding yellow fluorescent protein (YFP), a variant of green fluorescent protein, together with a neomycin resistance marker, and is driven by the SV40 promoter (eg SEQ ID NO:24). The second operon is the “business-end” of the vector for the purposes of this aspect of the invention, and comprises a nucleic acid sequence encoding a codon-optimized ScFv-FcγR fusion protein, comprising the hCMV-IE promoter and driven by the hCMV intron (eg SEQ ID NO:25).

CHO-K1 cells were transfected with the plasmid of SEQ ID NO:23. Stable components were isolated that integrated the linear structure of SEQ ID NO:22 into their genome.

The circular plasmid contains two Lox sites flanking the first operon and the second operon, allowing integration of the operon into the genome of the host cell as a linear structure. A linear structure spanning from a first Lox site to a second Lox site is exemplified in SEQ ID NO:22 and 5-prime to 3-prime SV40 promoter, nucleic acid encoding neomycin-resistance, IRES, eYFP Encoding nucleic acid, SV40 polyadenylation sequence, hCMV-IE promoter, hCMV intron, Tet-operator sequence (for controlled expression of ScFv-FcγR-TM-cyto fusion protein), nucleic acid encoding mROR signal sequence , a nucleic acid encoding the Ab2 ScFv, a nucleic acid encoding the FcγR transmembrane and cytoplasmic portion (SEQ ID NO: 21), and a SV40 polyadenylation sequence.

Example 17: ScFvs - FcγR - TM - cyto surface capture target

CHO-K1 cells containing the integrated sequence of SEQ ID NO:22 were prepared with antibodies of various subtypes, e.g., IgG1, IgG2, IgG4, containing one CH3 domain with 95R/435R-96F/436F double substitutions. Meanwhile, the other CH3 domain was transfected with a wild type IgG4 bispecific antibody (IgG4 Fc/Fc*), and a plasmid encoding an IgG1 bispecific antibody in an IgG1 Fc/Fc* format. Cells were treated with doxycycline to induce production of capture molecules along with antibodies. After co-expression of the antibody and capture molecule, in some cases cells were treated with an hFc blocking protein, and a detection molecule (FITC-labeled anti-hFab). Table 3 summarizes the results and shows that, in general, the ScFv-FcγR surface capture fusion proteins bind IgG4, IgG2, and IgG1 molecules, while the wild-type FcγR surface capture molecules bind IgG, but not IgG4 or IgG2. .

Blocking molecule competition assay Any FITC unit (with or without hFc blocking molecule) -mode hFc
how? antibody no hFc hFc (1 hour) hFc (2 hours) hFc (20 hours) uncoated Capture molecule = ScFv-FcγR-TM-cyto
Detection molecule = FITC-anti-hFab IgG1 mAb-3 250 120 80 20 10 yes IgG4 mAb-4 250 100 55 20 10 yes IgG4 mAb-5 250 70 40 20 10 yes IgG2 mAb-6 200 ¹ ND ND ND 12 ² yes Capture Molecule = hFcγR
Detection molecule = FITC-anti-hFab IgG1 mAb-3 300 80 30 9 3.5 yes IgG4 mAb-4 100 2 2 2 2 no IgG4 mAb-5 35 5 5 5 5 no

¹ + Dox ² - Dox

Example 18: Ab6 and Ab6 - originated ScFvs *- FcγR - TM - cyto cell line that produces

The heavy and light chains of Fc*-specific Ab6 were sequenced. The amino acid sequence of the light chain was determined to be SEQ ID NO:41. The amino acid sequence of the heavy chain was determined to be SEQ ID NO:40. To prepare the recombinant Ab6 antibody, an expression vector plasmid encoding the heavy chain was constructed and an expression vector plasmid encoding the light chain was constructed. To express the antibody, both plasmids were transfected into CHO-K1 cells, stable transformants were isolated, and expression was driven by the constitutive CMV promoter.

To prepare nucleic acids encoding Ab6-derived anti-Fc*-specific ScFv*-FcγR surface capture molecules, the immunoglobulin heavy chain variable domain of Ab6 antibody (SEQ ID NO:38) and the immunoglobulin light chain variable domain of Ab6 (SEQ ID NO:39) The amino acid sequence was reverse translated and codon optimized for CHO cell expression. Similarly, the C-terminal portion of human FcγRI (SEQ ID NO: 21) was codon optimized for CHO cell expression. The codon-optimized nucleotide sequence was amplified via polymerase chain reaction and ligated to form a contiguous nucleic acid sequence (SEQ ID NO:45) encoding an anti-Fc* ScFv*-FcγR fusion protein (SEQ ID NO:43).

A nucleic acid encoding the ScFv*-FcγR-TM-cyto fusion protein was inserted into an expression vector using standard PCR and restriction endonuclease cloning techniques. The resulting circular plasmid, exemplified by SEQ ID NO:44, contains a beta-lactamase-encoding nucleic acid sequence, and two operons. The first operon contains a nucleic acid sequence encoding yellow fluorescent protein (YFP), a variant of green fluorescent protein, together with a neomycin resistance marker, and is driven by the SV40 promoter (eg, SEQ ID NO:46). The second operon is the "business-end" of the vector for the purposes of this aspect of the invention, and comprises a nucleic acid sequence encoding a codon-optimized anti-Fc* ScFv-FcγR fusion protein, comprising the hCMV-IE promoter and hCMV Driven by an intron (eg SEQ ID NO:47).

CHO-K1 cells were transfected with the plasmid of SEQ ID NO:44. A stable component incorporating the linear structure of SEQ ID NO:48 was isolated.

The circular plasmid contains two Lox sites flanking a first operon and a second operon, allowing integration of the operon into the host cell's genome as a linear structure. A linear structure spanning a first Lox site to a second Lox site is exemplified in SEQ ID NO:48 and 5-prime to 3-prime SV40 promoter, nucleic acid encoding neomycin-resistance, IRES, nucleic acid encoding eYFP , SV40 polyadenylation sequence, hCMV-IE promoter, hCMV intron, Tet-operator sequence (for controlled expression of anti-Fc* ScFv*-FcγR fusion protein), nucleic acid encoding mROR signal sequence, Ab6-derived A nucleic acid encoding an anti-Fc*-specific ScFv*, a nucleic acid encoding the FcγR transmembrane and cytoplasmic portion (SEQ ID NO: 21), and a SV40 polyadenylation sequence.

Example 19: Bispecific antibody classification

port- Fc capture & port- Fc * detection

The Ab2-derived anti-Fc-specific ScFv-FcγR surface capture system was tested for its ability to detect and enrich cells testing the dual specific antibody. To evaluate the ability to detect bispecific antibodies with 95R/435R-96F/436F substitutions in one of the CH3 domains (designated as Fc*), various antibodies were tested with hFc as a blocking molecule and FITC-labeled as a detection molecule. Ab2-derived anti-Fc-specific ScFv-FcγR surface capture using an Ab6 anti-Fc* antibody (e.g., a mAb with HC of SEQ ID NO:40, LC of SEQ ID NO:41) expressed in cell lines. The Ab2-derived anti-Fc-specific ScFv-FcγR surface capture cell line uses an Fc*-specific Ab6 as a detection molecule to detect bispecific antibodies (Fc /Fc*) could be detected and distinguished (Table 4). The wild-type FcγR surface capture cell line was indistinguishable between the Fc/Fc*, Fc*/Fc*, and Fc/Fc IgG4 strains because FcγR either cannot bind IgG4 or binds with very low affinity.

port- Fc * capture & port- Fc detection

Conversely, the Ab6-derived anti-Fc*-specific ScFv*-FcγR surface capture system was tested for its ability to detect and enrich cells producing bispecific antibodies. To evaluate the ability to detect bispecific antibodies that have a 95R/435R-96F/436F substitution in one of the CH3 domains (designated as Fc*), various antibodies were tested with hFc as a blocking molecule and non-substituted as a detection molecule. using an Alexa 488-labeled Ab2 anti-Fc antibody recognizing CH3, expressed in an Ab6-derived anti-Fc*-specific ScFv*-FcγR surface capture cell line. The Ab6-derived anti-Fc*-specific ScFv*-FcγR surface capture cell line uses an Fc-specific Ab2 as a detection molecule to detect bispecific antibodies (better than any Fc*/Fc* or Fc/Fc monospecific antibodies). Fc/Fc*) could be detected and distinguished (Table 4). FcγR surface capture cell lines were indistinguishable between the Fc/Fc*, Fc*/Fc*, and Fc/Fc IgG4 species.

Detection of Bispecific Antibodies - Mean Fluorescence Intensity (MFI) IgG1 IgG4 ¹ CSCP ² DM Fc/Fc* Fc*/Fc* Fc/Fc Fc/Fc* Fc*/Fc* Fc/Fc Fc/Fc*
specificity FcgR Ab2 500 ND 350 200 200 200 no Ab6 200 200 200 ND ND ND no anti-hFc 1800 ND 1000 ND ND ND no ScFv-FcγR Ab6 500 15 15 500 15 15 yes anti-hFc ND ND ND ND ND ND ND ScFv*-FcγR Ab2 150 10 10 ND ND ND yes anti-hFc 200 ND 10 ND ND ND yes

¹ cell surface capture protein ² detection molecule

Example 20: Enrichment of Fc/Fc* bispecific antibodies

(Ab2-derived) ScFv-FcγR CSCP/ (Ab6) anti-Fc* DM and (Ab6-derived) ScFv*-FcγR CSCP/ (Ab2) anti-Fc DM systems to sort and enrich bispecific antibodies To evaluate the ability, Fc/Fc* IgG4 monoclonal antibody (IgG4-mAb-2) and anti-Fc ScFv were used, using hFc as blocking molecule and FITC-labeled anti-Fc* (Ab6) antibody as detection molecule. Cell lines co-expressing the -FcγR fusion protein were subjected to continuous fluorescence activated cell sorting and pooling to enrich for production of Fc/Fc* species. Cells harvesting Fc/Fc* from pools 5 and 7 were analyzed for total antibody titer and titer of each antibody format: Fc/Fc*, Fc/Fc, and Fc*/Fc*. Heavy chain encoding an unsubstituted CH3 domain ("Fc", i.e., histidine at IMGT position 95 and tyrosine at IMGT position 96) and substituted CH3 Since it encodes both heavy chains encoding domains ("Fc*", i.e., with arginine at IMGT position 95 and phenylalanine 96 at IMGT position 96), the cell could theoretically contain 25% Fc/Fc, 50% Fc/Fc*, and 25% Fc*/Fc*. Biologically, however, the majority (pre-enrichment) of antibodies produced would be expected to be Fc/Fc.

As shown in Table 5, cells selected, pooled, and enriched for bispecific antibody production produced as much as 49% Fc/Fc* species and at least about 3.2 g/L of Fc/Fc* bispecific antibody. have antibody titers.

Enrichment of the Fc/Fc* bispecific antibody IgG4-mAb-2 Fc/Fc* Fc/Fc Fc*/Fc* grass cell line Titer (g/L) % Titer (g/L) % Titer (g/L) % 5 One 1.2 28 2.2 50 0.99 23 2 1.9 49 1.3 32 0.73 19 3 1.5 47 1.2 40 0.40 13 4 1.6 37 1.3 31 1.3 32 5 1.5 48 1.1 35 0.58 18 6 1.8 47 1.3 33 0.75 20 6 7 2.6 44 2.0 34 1.3 23 8 3.2 42 2.4 31 2.0 27 9 2.1 45 1.5 33 1.0 22 10 2.8 43 2.0 31 1.7 28 11 2.3 44 1.6 31 1.3 24

Although the foregoing invention has been described in some detail by way of illustration and example, it will be readily apparent to those skilled in the art that certain changes and modifications may be made to the teachings of this invention without departing from the spirit or scope of the appended claims.

SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. <120> RECOMBINANT CELL SURFACE CAPTURE PROTEINS <130> 8600-WO <150> US 61/726,040 <151> 2012-11-14 <160> 51 <170> PatentIn version 3.5 <210> 1 <211> 195 <212> PRT <213> Streptococcus <400> 1 Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr 1 5 10 15 Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr 20 25 30 Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr 35 40 45 Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp Ala Ser Glu 50 55 60 Leu Thr Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr 65 70 75 80 Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu 85 90 95 Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp 100 105 110 Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu 115 120 125 Val Ile Asp Ala Ser Glu Leu Thr Pro Ala Val Thr Thr Tyr Lys Leu 130 135 140 Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Thr Lys Ala Val 145 150 155 160 Asp Ala Glu Thr Ala Glu Lys Ala Phe Lys Gln Tyr Ala Asn Asp Asn 165 170 175 Gly Val Asp Gly Val Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr 180 185 190 Val Thr Glu 195 <210> 2 <211 > 96 <212> PRT <213> Homo sapiens <400> 2 Gln Val Leu Gly Leu Gln Leu Pro Thr Pro Val Trp Phe His Val Leu 1 5 10 15 Phe Tyr Leu Ala Val Gly Ile Met Phe Leu Val Asn Thr Val Leu Trp 20 25 30 Val Thr Ile Arg Lys Glu Leu Lys Arg Lys Lys Lys Trp Asp Leu Glu 35 40 45 Ile Ser Leu Asp Ser Gly His Glu Lys Lys Val Thr Ser Ser Leu Gln 50 55 60 Glu Asp Arg His Leu Glu Glu Glu Leu Lys Cys Gln Glu Gln Lys Glu 65 70 75 80 Glu Gln Leu Gln Glu Gly Val His Arg Lys Glu Pro Gln Gly Ala Thr 85 90 95 <210> 3 <211> 33 <212> DNA <213> artificial sequence <220 > <223> synthetic <400> 3 cgggctgatg ctgcaccaac tgtatccatc ttc 33 <210> 4 <211> 33 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 4 acactctccc ctgttgaagc tcttgacaat ggg 33 <210> 5 <211> 31 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 5 gccaaaacaa cagccccatc ggtctatcca c 31 <210> 6 <211> 35 <212> DNA <213> artificial sequence <220> <223> Synthetic <400> 6 Tcattaccc GGAGTCCGGCTT AGAAGCTT AGAGCTT AGAGCTT AGAGCTT 35 <210> 7 <211> 47 <212> DNA <213> Artificial sequence <220> ACCT GCGTCATGCA GATGTGAAC TGCAGTC TGGGCCT 47 <210> 8 <211> 38 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 8 gagagacctg cgtcagctga ggagacggtg accgtggt 38 <210> 9 <211> 35 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 9 gagagggtct cacagccaaa acaacagccc catcg 35 <210> 10 <211> 42 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 10 gagagggtct ccggccgctc atttacccgg agtccgggag aa 42 <210> 11 <211> 40 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 11 gagagcgtct catgcagaca tccagatgac ccagtctcca 40 <210> 12 <211> 40 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 12 gagagcgtct cacagcccgt tttatttcca gcttggtccc 40 <210> 13 <211> 36 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 13 gagagggtct cagctgatgc tgcaccaact gtatcc 36 <210 > 14 <211> 48 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 14 gagagggtct caggccgctc aacactctcc cctgttgaag ctcttgac 48 <210> 15 <211> 113 <212> PRT <213> Homo sapiens <400> 15 Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala Ser Val 1 5 10 15 Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Trp Ile 20 25 30 His Trp Glu Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Tyr 35 40 45 Ile Asn Pro Asn Thr Gly His Thr Glu Tyr Asn Gln Lys Phe Lys Asp 50 55 60 Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr Met Gln 65 70 75 80 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 85 90 95 Thr Tyr Ser Gly Ser Ser His Phe Asp Tyr Trp Gly Gln Gly Thr Thr Thr 100 105 110 Leu <210> 16 <211> 112 <212> PRT < 213> Homo sapiens <400> 16 Ser Asp Ile Val Met Thr Gln Thr Pro Val Ser Leu Pro Val Ser Leu 1 5 10 15 Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His 20 25 30 Asn Asn Gly Asp Thr Phe Leu His Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln 85 90 95 Thr Thr Leu Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 <210> 17 <211> 21 <212> PRT <213> Homo sapiens <400> 17 Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu Val Asn Thr Val 1 5 10 15 Leu Trp Val Thr Ile 20 <210> 18 <211> 61 <212 > PRT <213> Homo sapiens <400> 18 Arg Lys Glu Leu Lys Arg Lys Lys Lys Trp Asp Leu Glu Ile Ser Leu 1 5 10 15 Asp Ser Gly His Glu Lys Lys Val Thr Ser Ser Leu Gln Glu Asp Arg 20 25 30 His Leu Glu Glu Glu Glu Leu Lys Cys Gln Glu Gln Lys Glu Glu Gln Leu 35 40 45 Gln Glu Gly Val His Arg Lys Glu Pro Gln Gly Ala Thr 50 55 60 <210> 19 <211> 334 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 19 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Glu Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Thr Gly His Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Thr Tyr Ser Gly Ser Ser His Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Ile Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Val Ser 130 135 140 Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 145 150 155 160 Gln Ser Leu Val His Asn Asn Gly Asp Thr Phe Leu His Trp Tyr Leu 165 170 175 Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn 180 185 190 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val 210 215 220 Tyr Phe Cys Ser Gln Thr Thr Leu Ile Pro Arg Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Val Leu Phe Tyr 245 250 255 Leu Ala Val Gly Ile Met Phe Leu Val Asn Thr Val Leu Trp Val Thr 260 265 270 Ile Arg Lys Glu Leu Lys Arg Lys Lys Lys Trp Asp Leu Glu Ile Ser 275 280 285 Leu Asp Ser Gly His Glu Lys Lys Val Thr Ser Ser Leu Gln Glu Asp 290 295 300 Arg His Leu Glu Glu Glu Leu Lys Cys Gln Glu Gln Lys Glu Glu Gln 305 310 315 320 Leu Gln Glu Gly Val His Arg Lys Glu Pro Gln Gly Ala Thr 325 330 <210> 20 <211> 1005 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 20 caagtacaac tgcaacaaag cggagctgaa ctggccaaac caggcgcttc cgtgaagatg 60 tct tgtaaag ccagcgggta tacatttaact aattactgga ttcactggga gaagcaaaga 120 cctgaacagg gattggaatg gattggatac attaatccta acaccggaca cacagagtat 180 aatcaaaaat tcaagtaa ggccaccctc acagccgaca gatcttcttc aaccgcctat 240 atgcaacttt cttcc ctcac ttctgaagac tccgcagttt actttgcgc acgaacttat 300 tctggaagct cccatttcga ctactggggt caaggaacaa cactgatcgt gtctagcggc 360 ggcggagggt ccggcggggg cggtagcggt ggcggaggtt ctgatattgt catgactcaa 420 acacct gtct ctctgcctgt ttcacttgga gatcaagcta gcatttcctg ccgctctagt 480 caatctctcg tccacaacaa cggcgatact ttcttgcatt ggtatctgca gaaaccaggt 540 cagtcaccta aactgcttat atacaaagtc tctaatagat tctcaggggt gccagatcga 600 ttcagtggtt ctgggtccgg tacagatttt acactcaaga tatccagagt agaagcagaa 660 gatctgggcg t a aaaaatggg atctggaaat atcattggac agtggacacg aaaaaaaagt cacatcatca 900 ttgcaagaag accggcactt ggaggagggaa ctgaaatgtc aagagcaaaa agaagaacaa 960 ctgcaagaag gcgtacatag aaaagaacca cagggagcaa catag 1005 <210> 21 <211> 82 <212> PRT <213> Homo sapiens <400> 21 Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu Val Asn Thr Val 1 5 10 15 Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys Lys Lys Lys Trp Asp 20 25 30 Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys Val Thr Ser Ser 35 40 45 Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys Cys Gln Glu Gln 50 55 60 Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys Glu Pro Gln Gly 65 70 75 80 Ala Thr <210> 22 <211> 5759 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 22 acaacttcgt atagcataca ttatacgaag ttatggtacc aagcctaggc ctccaaaaaa 60 gcctcctcac tacttctgga atagctcaga ggcagaggcg gcctcggcct ctgcataaat 120 aaaaaaaatt agtcagccat ggggcggaga atgggcg gaa ctgggcggag ttaggggcgg 180 gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca tgctttgcat 240 acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg 300 catgctttgc atacttctgc ct gctgggga gcctggggac tttccacacc ggatccacca 360 tgggttcagc tattgagcag gatgggttgc atgctggtag tcccgccgca tgggtcgaac 420 gactgtttgg atacgattgg gcccaacaga ctataggctg ttccgacgct gctgtctttc 480 gtctttctgc acaaggtcgt ccagttctgt tcgtgaaaac cgacttgtcc ggagccctca 540 atgagttgca agacgaagct gcacgactga gttggcttgc caccactggt gtcccatgtg 600 ccgcagtact tgacgtcgtc acagaggctg gtcgcgattg gttgctcctt ggagaagtgc 660 ccggccaaga tcttctcagt tcccaccttg cccctgcc ga aaaagtttca ataatggctg 720 acgctatgag aaggctgcac acccttgacc ctgccacatg tccattcgat caccaagcca 780 aacaccgaat aacataat gg 960 tagagaatgg aagatttagc ggcttcattg attgtggacg acttggagtt gcagatcggt 1020 accaagatat cgctctcgct accagagata ttgctgaaga attgggcgga gaatgggctg 1080 atcggtttct cgtactctac ggaattgccg cacctgattc ccaacgcatt gctttttacc 1140 gtcttctgga tgagttcttc taaacgcgtc ccccctctcc ctcccccccc cctaacgtta 1200 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca 1260 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca 1320 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgt gaagg 1380 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc 1440 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata 1500 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag 1560 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc 1620 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt 1680 taaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatt 1740 gctcgaatca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 1800 gtcgagctgg acggcg acgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 1860 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 1920 ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc 198 0 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 2040 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 2100 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 2160 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 2220 aagcagaaga acggcatcaa ggtgaacttc aagatccg cc acaacatcga ggacggcagc 2280 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 2340 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc 2400 gatcacatgg tcctgctgga gttcgtga cc gccgccggga tcactctcgg catggacgag 2460 ctgtacaagt aatcggccgc taatcagcca taccacattt gtagaggttt tacttgcttt 2520 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt 2580 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 2640 aaataaagca tttttttcac tgcatt acc gcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 2880 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 2940 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 3000 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 3060 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggtt t tggcagtaca 3120 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 3180 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 3240 ccgccccatt gacgcaaatg ggcggtag gc gtgtacggtg ggaggtctat ataagcagag 3300 ctctccctat cagtgataga gatctcccta tcagtgatag agatcgtcga cgtttagtga 3360 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg 3420 accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga 3480 gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttct tatgcatgct 354 0 atactgtttt tggcttgggg tctatacacc cccgcttcct catgttatag gtgatggtat 3600 agcttagcct ataggtgtgg gttattgacc attattgacc actcccctat tggtgacgat 3660 actttccatt actaatccat aacatggctc tttgccacaa ctctctttat tggctatatg 37 20 ccaatacact gtccttcaga gactgacacg gactctgtat ttttacagga tggggtctca 3780 tttatattt acaaattcac atatacaaca ccaccgtccc cagtgcccgc agtttttatt 3840 aaacataacg tgggatctcc acgcgaatct cgggtacgtg ttccggacat ggtctcttct 3900 ccggtagcgg cggagcttct acatccgagc cctgctccca tgcctccagc gactcatggt 3960 cgctcggcag ctccttgctc ctaacagtgg aggccagact taggcacagc acgatgccca 4020 ccaccaccag tgtgccgcac aaggccgtgg cggtagggta tgtgtctgaa aatgagctcg 4080 gggagcgggc ttgcaccgct gacgcatttg gaagacttaa ggcagcggca ga agaagatg 4140 caggcagctg agttgttgtg ttctgataag agtcagaggt aactcccgtt gcggtgctgt 4200 taacggtgga gggcagtgta gtctgagcag tactcgttgc tgccgcgcgc gccaccagac 4260 ataatagctg acagactaac agactgttcc tttccatggg tcttttctgc agtcaccgtc 4320 cttgacacga agcttatact cgagctctag attgggaacc cgggtctctc gaattcgaga 438 0 tctccaccat gcacagacct agacgtcgtg gaactcgtcc acctccactg gcactgctcg 4440 ctgctctcct cctggctgca cgtggtgctg atgcacaagt acaactgcaa caagcggag 4500 ctgaactggc caaaccaggc gcttccgtga agatgtcttg taaagccagc gggtata cat 4560 ttactaatta ctggattcac tgggaagaagc aaagacctga acagggattg gaatggattg 4620 gatacattaa tcctaacacc ggacacacag agtataatca aaaattcaag gataaggcca 4680 ccctcacagc cgacagatct tcttcaaccg cctatatgca actttcttcc ctcacttctg 4740 aagactccgc agtttacttt tgcgcacgaa cctattctgg aagctcccat ttcgactact 4800 ggggtcaagg aacaacact g atcgtgtcta gcggcggcgg agggtccggc gggggcggta 4860 gcggtggcgg aggttctgat attgtcatga ctcaaacacc tgtctctctg cctgtttcac 4920 ttggagatca agctagcatt tcctgccgct ctagtcaatc tctcgtccac aacaacggcg 4980 atactt tctt gcattggtat ctgcagaaac caggtcagtc acctaaactg cttatataca 5040 aagtctctaa tagattctca ggggtgccag atcgattcag tggttctggg tccggtacag 5100 attttacact caagatatcc agagtagaag cagaagatct gggcgtgtat ttctgcagtc 5160 aaacaacact tattcctcgt acttttggag gcggtacaaa actggagatc aagcgtggag 5220 gcggagggag tgttttgttt tatctggccg ttgggataat gtttctcgta aatacagtac 5280 tttgggtaac aataaggaag gaactgaaga gaaagaaaaa atgggatctg gaaatatcat 5340 tggacagtgg acacgaaaaa aaagtcacat catcattgca agaagaccgg cacttggagg 5400 aggaactgaa atgtcaagag caaa aagaag aacaactgca agaaggcgta catagaaaag 5460 aaccacaggg agcaacatag gcggccgcta atcagccata ccacatttgt agaggtttta 5520 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt 5580 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 5640 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 5700 aatgtatctt atcatgtcta ccggtataac ttcgtataat gtatactata cgaagttag 5759 <210> 23 <211> 7627 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 23 aagctttatac tcgagctcta gattgggaac ccgggtctct cgaattcgag atctccacca 60 tgcacagacc tagacgtcgt ggaactcgtc cacctccact ggcactgctc gctgctctcc 120 tcctggctgc acgtggtgct gatgcacaag tacaactgca acaaag cgga gctgaactgg 180 ccaaaccagg cgcttccgtg aagatgtctt gtaaagccag cgggtataca tttactaatt 240 actggattca ctgggagaag caaagacctg aacagggatt ggaatggatt ggatacatta 300 atcctaacac cggacacaca gagtataatc aaaaattcaa ggataaggcc accctcacag 360 ccgacagatc ttcttcaacc gcctatatgc aactttcttc cctcacttct gaagactccg 420 cagtttactt ttgcgcacga acttattctg gaagctccca tttcgactac tggggtcaag 480 gaacaacact gatcgtgtct agcggcggcg gagggtccgg cgggggcggt agcggtggcg 540 gaggttctga tattgtcatg actcaaacac ctgtctctct gcctgtttca cttggagatc 600 aagctagcat ttcctgccgc tctagtcaat ctctcgtcca caacaacggc gatactttct 660 tgcattggta tctgcagaaa ccaggtcagt cacctaaact gcttatatac aaagtctcta 720 atagattctc aggggtgcca gatcgattca gtggttctgg gtccggtaca gattttacac 78 0 tcaagatatc cagagtagaa gcagaagatc tgggcgtgta tttctgcagt caaacaacac 840 ttattcctcg tacttttgga ggcggtacaa aactggagat caagcgtgga ggcggaggga 900 gtgttttgtt ttatctggcc gttgggataa tgtttctcgt aaata cagta ctttgggtaa 960 caataaggaa ggaactgaag agaaagaaaa aatgggatct ggaaatatca ttggacagtg 1020 gacacgaaaa aaaagtcaca c ctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 1260 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 1320 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 1380 tatcatgtct accgg tataa cttcgtataa tgtatactat acgaagttag ccggtagggc 1440 ccctctcttc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 1500 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 1560 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 1620 cctcgtgcg c tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 1680 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 1740 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 1800 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 1860 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 1920 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 1980 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 2040 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 2100 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 2160 gattttggtc atgggcgcgc ctcatactcc tgcaggcatg agattatcaa aaaggatctt 2220 cacc tagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 2280 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 2340 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 2400 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 2460 tttatcagca ataaaccagc cagccggaag ggccgagcg c agaagtggtc ctgcaacttt 2520 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 2580 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 2640 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 2700 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc aatact gcgc cacatagcag 2940 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 3000 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 3060 ttttactttc accagcgttt ctgggt gagc aaaaacagga aggcaaaatg ccgcaaaaaa 3120 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 3180 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 3240 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcaggtacac 3300 aacttcgtat agcatacatt atacgaagtt atggtaccaa gcctaggcct ccaaaa aagc 3360 ctcctcacta cttctggaat agctcagagg cagaggcggc ctcggcctct gcataaataa 3420 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga 3480 tgggcggagt taggggcggg actatggttg ctgactaatt gaga tgcatg ctttgcatac 3540 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca 3600 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccgg atccaccatg 3660 ggttcagcta ttgagcagga tgggttgcat gctggtagtc ccgccgcatg ggtcgaacga 3720 ctgtttggat acgattgggc ccaacagact ataggctgtt ccgacgctgc tgtctttcgt 3780 ctttctgcac aaggtcgtcc agttctgttc gtgaaaaccg acttgtccgg agccctcaat 3840 gagttgcaag acgaagctgc acgactgagt tggcttgcca ccactggtgt cccatgtgcc 3900 gcagtacttg acgtcgtcac agaggctggt cgcgattggt tgctcc ttgg agaagtgccc 3960 ggccaagatc ttctcagttc ccaccttgcc cctgccgaaa aagtttcaat aatggctgac 4020 gctatgagaa ggctgcacac ccttgaccct gccacatgtc cattcgatca ccaagccaaa 4080 caccgaattg aacgagctag aacccgcatg gaagccggcc tcgttgatca agacgatttg 4140 gatgaggaac accagggtct cgcacccgct gaactcttcg ctcgcctcaa agcacgaatg 4200 ccaga cggag atgacttggt cgtaacccac ggagatgcct gccttcctaa cataatggta 4260 gagaatggaa gatttagcgg cttcattgat tgtggacgac ttggagttgc agatcggtac 4320 caagatatcg ctctcgctac cagagatatt gctgaagaat tgggcggaga atgggctgat 4380 cggtttctcg tactctacgg aattgccgca cctgattccc aacgcattgc tttttaccgt 4440 cttctggatg agttcttcta aacgcgtccc ccctctccct cccccccccc taacgttact 4500 ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata 4560 ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt 4620 cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa 4680 gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag 4740 cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca 4800 c ctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc 4860 aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat 4920 tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta 4980 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgattgc 5040 tcgaatcacc atggtgagca agggcga gga gctgttcacc ggggtggtgc ccatcctggt 5100 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga 5160 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc 5220 ctggcccacc ctcgtgacca ccttcggcta cggcctgcag tgcttcgccc gctaccccga 5280 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg 5340 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg 5400 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat 5460 cctggggcac aagctggagt acaactaca cagccaca c ctgagcaaa gaccccaacg agaagcgcga 5700 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct 5760 gtacaagtaa tcggccgcta atcagccata ccacatttgt agaggtttta cttgctttaa 5820 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta 5880 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aattt cacaa 5940 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 6000 atcatgtcgg cgcgttgaca ttgattattg actagttat aatagtaatc aattacgggg 6060 tcattagttc atagcccata tatggagttc cgcgttacat aacttacgg t aaatggcccg 6120 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 6180 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 6240 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 6300 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 6 360 cagtacatct acgttatagt catcgctatt accatggtga tgcggttttg gcagtacatc 6420 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 6480 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgt cg taacaactcc 6540 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 6600 ctccctatca gtgatagaga tctccctatc agtgatagag atcgtcgacg tttagtgaac 6660 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac 6720 cgatccagcc tccgcggccg ggaacggtgc attggaacgc ggattccccg tgccaagagt 6 780 gacgtaagta ccgcctatag agtctatagg cccaccccct tggcttctta tgcatgctat 6840 actgtttttg gcttggggtc tatacacccc cgcttcctca tgttataggt gatggtatag 6900 cttagcctat aggtgtgggt tattgaccat tattgaccac tcccctattg gtgacga tac 6960 tttccattac taatccataa catggctctt tgccacaact ctctttattg gctatatgcc 7020 aatacactgt ccttcagaga gag cttctac atccgagccc tgctcccatg cctccagcga ctcatggtcg 7260 ctcggcagct ccttgctcct aacagtggag gccagactta ggcacagcac gatgcccacc 7320 accaccagtg tgccgcacaa ggccgtggcg gtagggtatg tgtctgaaaa tgagctcggg 738 0 gagcgggctt gcaccgctga cgcatttgga agacttaagg cagcggcaga agaagatgca 7440 ggcagctgag ttgttgtgtt ctgataagag tcagaggtaa ctcccgttgc ggtgctgtta 7500 acggtggagg gcagtgtagt ctgagcagta ctcgttgctg ccgcgcgcgc caccagacat 7560 aatagctgac agactaacag actgttcctt tccatgggtc ttttctgcag tcaccgtcct 7620 t gacacg 7627 <210> 24 <211> 2669 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 24 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gcagaggcgg 60 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac 120 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat 180 tgagatgcat g ctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg 240 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact 300 ttccacaccg gatccaccat gggttcagct attgagcagg atgggttgca tgctggtagt 360 cccgccgcat gggtcgaacg actgtttgga tacgattggg cccaacagac tataggctgt 420 tccgacgctg ctgtctttcg tctttctgca caaggtcgtc cagttctgtt cgtgaaaacc 480 gacttgtccg gagccctcaa tgagttgcaa gacgaagctg cacgactgag ttggcttgcc 540 accactggtg tcccatgtgc cgcagtactt gacgtcgtca cagaggctgg tcgcgattgg 600 ttgctccttg gagaag tgcc cggccaagat cttctcagtt cccaccttgc ccctgccgaa 660 aaagtttcaa taatggctga cgctatgaga aggctgcaca cccttgaccc tgccacatgt 720 ccattcgatc accaagccaa acaccgaatt gaacgagcta gaacccgcat ggaagccggc 780 ctcgtt gatc aagacgattt ggatgaggaa caccagggtc tcgcacccgc tgaactcttc 840 gctcgcctca aagcacgaat gccagacgga gatgacttgg tcgtaaccca cggagatgcc 900 tgccttccta acataatggt agagaatgga agatttagcg gcttcattga ttgtggacga 960 cttggagttg cagatcggta ccaagatatc gctctcgcta ccagagatat tgctgaagaa 1020 ttgggcggag aatgggctga tc ggtttctc gtactctacg gaattgccgc acctgattcc 1080 caacgcattg ctttttaccg tcttctggat gagttcttct aaacgcgtcc cccctctccc 1140 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc 1200 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc 1260 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt 1320 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct 1380 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa 1440 aagccacgtg tataagatac acctgcaaag gc ggcacaac cccagtgcca cgttgtgagt 1500 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag 1560 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt 1620 acatgtgttt agtc gaggtt aaaaaacgtc taggcccccc gaaccacggg gacgtggttt 1680 tcctttgaaa aacacgattg ctcgaatcac catggtgagc aagggcgagg agctgttcac 1740 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 1800 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 1860 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc acct tcggct acggcctgca 1920 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 1980 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 2040 cgccgaggtg aagttcgagg gcgacacc ct ggtgaaccgc atcgagctga agggcatcga 2100 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 2160 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 2220 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 2280 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgag caa 2340 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 2400 cactctcggc atggacgagc tgtacaagta atcggccgct aatcagccat accacatttg 2460 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aa acataaaa 2520 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca 2580 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 2640 ccaaactcat caatgtatct tatcatgtc 2669 <210> 25 <211> 3003 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 25 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420 agcggtttga ctcacgggga tttccaagtc tcc accccat tgacgtcaat gggagtttgt 480 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg 600 atagagatct ccctatcagt gatagagatc gtcgacgttt agtgaaccgt cagatcgcct 660 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tcc agcctcc 720 gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac gtaagtaccg 780 cctatagagt ctataggccc acccccttgg cttcttatgc atgctatact gtttttggct 840 tggggtctat acacccccgc ttcctcatgt tataggtgat ggtatagct t agcctatagg 900 tgtgggttat tgaccattat tgaccactcc cctattggtg acgatacttt ccattactaa 960 tccataacat ggctctttgc cacaactctc tttattggct atatgccaat acactgtcct 1020 tcagagactg acacggactc tgtattttta caggatgggg tctcatttat tatttacaaa 1080 ttcacatata caacaccacc gtccccagtg cccgcagttt ttattaaaca taacgtggga 11 40 tctccacgcg aatctcgggt acgtgttccg gacatggtct cttctccggt agcggcggag 1200 cttctacatc cgagccctgc tcccatgcct ccagcgactc atggtcgctc ggcagctcct 1260 tgctcctaac agtggaggcc agacttaggc acagcacgat gcccacc acc accagtgtgc 1320 cgcacaaggc cgtggcggta gggtatgtgt ctgaaaatga gctcggggag cgggcttgca 1380 ccgctgacgc atttggaaga cttaaggcag cggcagaaga agatgcaggc agctgagttg 1440 ttgtgttctg ataagagtca gaggtaactc ccgttgcggt gctgttaacg gtggagggca 1500 gtgtagtctg agcagtactc gttgctgccg cgcgcgccac cagacataat agctgacaga 15 60 ctaacagact gttcctttcc atgggtcttt tctgcagtca ccgtccttga cacgaagctt 1620 atactcgagc tctagattgg gaacccgggt ctctcgaatt cgagatctcc accatgcaca 1680 gacctagacg tcgtggaact cgtccacctc cactggcact gctcgctgct ctcctcct gg 1740 ctgcacgtgg tgctgatgca caagtacaac tgcaacaaag cggagctgaa ctggccaaac 1800 caggcgcttc cgtgaagatg tcttgtaaag ccagcgggta tacatttact aattactgga 1860 ttcactggga gaagcaaaga cctgaacagg gattggaatg gattggatac attaatccta 1920 acaccggaca cacagagtat aatcaaaaat tcaagtaa ggccaccctc acagccgaca 1980 gatcttcttc aaccgccta t atgcaacttt cttccctcac ttctgaagac tccgcagttt 2040 acttttgcgc acgaacttat tctggaagct cccatttcga ctactggggt caaggaacaa 2100 cactgatcgt gtctagcggc ggcggagggt ccggcgggggg cggtagcggt ggcggaggtt 2160 ctgatattgt catgactcaa acacctgtct ctctgcctgt ttcacttgga gatcaagcta 2220 gcatttcctg ccgctctagt caatctctcg tccacaacaa cggcgatact ttcttgcatt 2280 ggtatctgca gaaaccaggt cagtcaccta aactgcttat atacaaagtc tctaatagat 2340 tctcaggggt gccagatcga ttcagtggtt ctgggtccgg tacagatttt acactcaaga 2400 tatccagagt agaagcagaa gatctgggcg tgtatttctg cagtcaaaca acacttattc 2460 ctcgtacttt tggaggcggt acaaaactgg agatcaagcg tggaggcgga gggagtgttt 2520 tgttttatct ggccgttggg ataatgtttc tcgtaaatac agtactttgg gtaacaataa 2580 ggaaggaact gaagagaaag aaaaaatggg atctggaaat atcattggac agtggacacg 2640 aaaaaaaagt cacatcatca ttgcaagaag accggcactt ggaggaggaa ctgaaatgtc 2700 aagagcaaaa agaagaacaa ctgcaagaag gcgtacatag aaaagaacca cagggagcaa 2760 cataggcggc cgctaatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac 2820 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg 2880 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 2940 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 3000 gtc 3003 <21 0> 26 <211> 208 <212 > PRT <213> Homo sapiens <400> 26 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 1 5 10 15 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 20 25 30 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 35 40 45 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 50 55 60 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 65 70 75 80 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 85 90 95 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 100 105 110 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 115 120 125 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 130 135 140 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 145 150 155 160 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 165 170 175 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 180 185 190 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 195 200 205 <210> 27 <211> 8 <212> PRT <213> Homo sapiens <400> 27 Gly Tyr Thr Phe Thr Asn Tyr Trp 1 5 <210> 28 <211> 8 <212> PRT <213> Homo sapiens <400> 28 Ile Asn Pro Asn Thr Gly His Thr 1 5 <210> 29 <211> 13 <212> PRT <213> Homo sapiens <400> 29 Cys Ala Arg Thr Tyr Ser Gly Ser Ser His Phe Asp Tyr 1 5 10 <210> 30 <211> 10 <212> PRT <213> Homo sapiens <400> 30 Ser Leu Val His Asn Asn Gly Asp Thr Phe 1 5 10 <210 > 31 <211> 9 <212> PRT <213> Homo sapiens <400> 31 Ser Gln Thr Thr Leu Ile Pro Arg Thr 1 5 <210> 32 <211> 8 <212> PRT <213> Homo sapiens <400> 32 Gly Phe Thr Phe Ser Asn Ala Trp 1 5 <210> 33 <211> 10 <212> PRT <213> Homo sapiens <400> 33 Ile Leu Ser Lys Thr Asp Gly Gly Thr Thr 1 5 10 <210> 34 < 211> 13 <212> PRT <213> Homo sapiens <400> 34 Thr Thr Ala Asp Phe Trp Ser Ala Tyr Ser Ser Asp Tyr 1 5 10 <210> 35 <211> 4 <212> PRT <213> Homo sapiens < 400> 35 Gln Ser Leu Leu 1 <210> 36 <211> 7 <212> PRT <213> Homo sapiens <400> 36 His Ser Asn Gly Tyr Asn Tyr 1 5 <210> 37 <211> 9 <212> PRT <213> Homo sapiens <400> 37 Met Gln Gly Leu Gln Thr Pro Tyr Thr 1 5 <210> 38 <211> 122 <212> PRT <213> Homo sapiens <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Ala Ile Val Lys Pro Gly Gly 1 5 10 15 Ser His Arg Val Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Leu Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Met 65 70 75 80 Leu Phe Leu Gln Met Asp Ser Leu Lys Ile Glu Asp Thr Ala Val Tyr 85 90 95 Phe Cys Thr Thr Ala Asp Phe Trp Ser Ala Tyr Ser Ser Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 112 <212> PRT <213> Homo sapiens <400> 39 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Met Glu Ala Glu Asp Val Gly Val Tyr Cys Met Gln Gly 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 40 <211 > 452 <212> PRT <213> Homo sapiens <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Ala Ile Val Lys Pro Gly Gly 1 5 10 15 Ser His Arg Val Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Leu Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Met 65 70 75 80 Leu Phe Leu Gln Met Asp Ser Leu Lys Ile Glu Asp Thr Ala Val Tyr 85 90 95 Phe Cys Thr Thr Ala Asp Phe Trp Ser Ala Tyr Ser Ser Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Ser Val Thr Val 180 185 190 Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro 210 215 220 Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 245 250 255 Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 275 280 285 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 290 295 300 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 305 310 315 320 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro 325 330 335 Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln 340 345 350 Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val 355 360 365 Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val 370 375 380 Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 405 410 415 Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val 420 425 430 Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg 435 440 445 Thr Pro Gly Lys 450 <210> 41 <211> 219 <212> PRT <213> Homo sapiens <400> 41 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Met Glu Ala Glu Asp Val Gly Val Tyr Cys Met Gln Gly 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 42 <211> 256 <212> PRT <213> Homo sapiens <400> 42 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Gly Pro Gly Asp Lys Thr 20 25 30 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 35 40 45 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 50 55 60 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 65 70 75 80 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 85 90 95 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 100 105 110 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 115 120 125 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 130 135 140 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 145 150 155 160 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 165 170 175 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 180 185 190 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 195 200 205 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 210 215 220 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 225 230 235 240 Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 250 255 <210> 43 <211> 336 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Ala Ile Val Lys Pro Gly Gly 1 5 10 15 Ser His Arg Val Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Leu Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Met 65 70 75 80 Leu Phe Leu Gln Met Asp Ser Leu Lys Ile Glu Asp Thr Ala Val Tyr 85 90 95 Phe Cys Thr Thr Ala Asp Phe Trp Ser Ala Tyr Ser Ser Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser 130 135 140 Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys 145 150 155 160 Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp 165 170 175 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu 180 185 190 Gly Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Met Glu Ala Glu Asp 210 215 220 Val Gly Val Tyr Tyr Cys Met Gln Gly Leu Gln Thr Pro Tyr Thr Phe 225 230 235 240 Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Val Leu 245 250 255 Phe Tyr Leu Ala Val Gly Ile Met Phe Leu Val Asn Thr Val Leu Trp 260 265 270 Val Thr Ile Arg Lys Glu Leu Lys Arg Lys Lys Lys Trp Asp Leu Glu 275 280 285 Ile Ser Leu Asp Ser Gly His Glu Lys Lys Val Thr Ser Ser Leu Gln 290 295 300 Glu Asp Arg His Leu Glu Glu Glu Leu Lys Cys Gln Glu Gln Lys Glu 305 310 315 320 Glu Gln Leu Gln Glu Gly Val His Arg Lys Glu Pro Gln Gly Ala Thr 325 330 335 <210> 44 <211> 7633 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 44 aagcttatac tcgagctcta gattgggaac ccgggtctct cgaattcgag atctccacca 60 tgcacagacc tagacgtcgt ggaactcgtc cacct ccact ggcactgctc gctgctctcc 120 tcctggctgc acgtggtgct gatgcagagg tgcagctggt ggagtctggg ggagccatag 180 taaagccggg ggggtcccat agagtctcct gtgaagcctc tggattcact ttcagtaacg 240 cctggatgag ttgggtccgc caggctccag ggagggggct ggaggtgggtt ggccgtattt 300 taagcaagac tgatggtggg acgacagact acgctgcacc cgtga aagac agattcacca 360 tttcaagaga tgattctaaa aatatgttgt ttctgcaaat ggacagcctg aaaatcgagg 420 acacagccgt gtatttctgt accacggccg attttggag tgcttattct tctgactact 480 ggggccaggg aaccctggtc accgtctcct caggaggt gg aggttccggg ggcgggggct 540 ccggcggagg tggatcagat attgtgatga ctcagtctcc actctccctg cccgtcaccc 600 ctggagagcc ggcctccatc tcctgcaggt ctagtcagag cctcctgcat agtaatgggt 660 acaactattt ggattggtac ctacagaagc cagggcagtc tccacaactc ctgatctatt 720 tgggttctaa tcgggcctcc ggggtccctg acaggttcag tggcagg ga tcaggcacag 780 attttacact gaaaatcagc agaatggagg ctgaggatgt tggggtttat tactgcatgc 840 aaggtctaca aactccgtac acttttggcc aggggaccaa gctggagatc aaaggaggcg 900 gagggagtgt tttgttttat ctggccgttg ggataatgtt t ctcgtaaat acagtacttt 960 gggtaacaat aaggaaggaa ctgaagagaa agaaaaaatg ggatctggaa atatcattgg 1020 acagtggaca cgaaaaaaaa gtcacatcat cattgcaaga agaccggcac ttggaggagg 1080 aactgaaatg tcaagagcaa aaagaagaac aactgcaaga aggcgtacat agaaaagaac 1140 cacagggagc aacataggcg gccgctaatc agccatacca catttgtaga ggttttactt 12 00 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt 1260 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 1320 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtcca a actcatcaat 1380 gtatcttatc atgtctaccg gtataacttc gtataatgta tactatacga agttagccgg 1440 tagggcccct ctcttcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 1500 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 1560 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 1620 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 1680 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 1740 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc c ccgttcagc ccgaccgctg 1800 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 1860 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 1920 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 1980 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 2040 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 2100 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 2160 ttaagggatt ttggtcatgg gcgcgcctca tactcctgca ggcatgaga t tatcaaaaag 2220 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 2280 tgagtaaact tggtctgaca gttaccaatg accag ccagc cggaagggcc gagcgcagaa gtggtcctgc 2520 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 2580 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 2640 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 2700 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 2760 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 2820 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 2880 gtgtatgcgg cgaccgagtt gctcttg ccc ggcgtcaata cgggataata ctgcgccaca 2940 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 3000 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 3060 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 3120 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 3180 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 3240 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcag 3300 gtacacaact tcgtatagca tacattatac gaagttat gg taccaagcct aggcctccaa 3360 aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg gcctctgcat 3420 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg 3480 gcgggatggg cggagttagg ggcggg acta tggttgctga ctaattgaga tgcatgcttt 3540 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga 3600 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccggatcc 3660 accatgggtt cagctattga gcaggatggg ttgcatgctg gtagtcccgc cgcatgggtc 3720 gaacgactgt ttggatacga ttgggcccaa cagactatag gctgtt ccga cgctgctgtc 3780 tttcgtcttt ctgcacaagg tcgtccagtt ctgttcgtga aaaccgactt gtccggagcc 3840 ctcaatgagt tgcaagacga agctgcacga ctgagttggc ttgccacac tggtgtccca 3900 tgtgccgcag tacttg acgt cgtcacagag gctggtcgcg attggttgct ccttggagaa 3960 gtgcccggcc aagatcttct cagttcccac cttgcccctg ccgaaaaagt ttcaataatg 4020 gctgacgcta tgagaaggct gcacaccctt gaccctgcca catgtccatt cgatcaccaa 4080 gccaaacacc gaattgaacg agctagaacc cgcatggaag ccggcctcgt tgatcaagac 4140 gatttggatg aggaacacca gggtctcgca cccgctgaac tcttcgctcg cctcaaagca 4200 cgaatgccag acggagatga cttggtcgta acccacggag atgcctgcct tcc taacata 4260 atggtagaga atggaagatt tagcggcttc attgattgtg gacgacttgg agttgcagat 4320 cggtaccaag atatcgctct cgctaccaga gatattgctg aagaattggg cggagaatgg 4380 gctgatcggt ttctcgtact ctacggaatt gccgcacctg attcccaacg cattgct ttt 4440 taccgtcttc tggatgagtt cttctaaacg cgtcccccct ctccctcccc cccccctaac 4500 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 4560 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 4620 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtc ggccac gttg tgagttggat agttgtggaa 4860 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 4920 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg 4980 aggttaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac 5040 gattgctcga atcaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat 5100 cctgg tcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga 5160 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccacg gcaagctgcc 5220 cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgcagtgct tc gcccgcta 5280 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca 5340 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt 5400 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg 5460 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc 5520 cgacaagcag aagaacggca tcaaggtga a cttcaagatc cgccacaaca tcgaggacgg 5580 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct 5640 gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa 5700 gcgcgatcac atggt cctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga 5760 cgagctgtac aagtaatcgg ccgctaatca gccataccac atttgtagag gttttacttg 5820 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg 5880 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 5940 tcacaaataa agcatttttt tcactgcat t ctagttgtgg tttgtccaaa ctcatcaatg 6000 tatcttatca tgtcggcgcg ttgacattga ttatgacta gttattaata gtaatcaatt 6060 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 6120 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 6180 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 6240 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 6300 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 6360 acttggcagt acatctacgt attagtcatc gctattacca tggt gatgcg gttttggcag 6420 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 6480 gacgtcaatg ggaggtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 6540 aactccgccc cattgacgca aatggg cggt aggcgtgtac ggtgggaggt ctatataagc 6600 agagctctcc ctatcagtga tagagatctc cctatcagtg atagatcg tcgacgttta 6780 aagagtgacg taagtaccgc ctatagagtc tataggccca cccc cttggc ttcttatgca 6840 tgctatactg tttttggctt ggggtctata cacccccgct tcctcatgtt ataggtgatg 6900 gtatagctta gcctataggt gtgggttatt gaccattatt gaccactccc ctattggtga 6960 cgatactttc cattactaat ccataacatg gctcttt gcc acaactctct ttattggcta 7020 tatgccaata cactgtcctt cagagactga cacggactct gtatttttac aggatggggt 7080 ctcatttatt atttacaaat tcacatatac aacaccaccg tccccagtgc ccgcagtttt 7140 tattaaacat aacgtgggat ctccacgcga atctcgggta cgtgttccgg acatggtctc 7200 ttctccggta gcggcggagc ttctacatcc gagccctgct cccatgcctc cagc gactca 7260 tggtcgctcg gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacgatg 7320 cccaccacca ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag 7380 ctcggggagc gggcttgcac cgctgacgca tttg gaagac ttaaggcagc ggcagaagaa 7440 gatgcaggca gctgagttgt tgtgttctga taagagtcag aggtaactcc cgttgcggtg 7500 ctgttaacgg tggagggcag tgtagtctga gcagtactcg ttgctgccgc gcgcgccacc 7560 agacataata gctgacagac taacagactg ttcctttcca tgggtctttt ctgcagtcac 7620 cgtccttgac acg 7633 <210> 45 <211> 1011 <212 > DNA <213> Artificial sequence <220> <223> synthetic <400> 45 gaggtgcagc tggtggagtc tgggggagcc atagtaaagc cgggggggtc ccatagagtc 60 tcctgtgaag cctctggatt cactttcagt aacgcctgga tgagttgggt ccgccaggct 120 ccagggaggg ggctggagtg ggttggccgt attttaagca agactgatgg tgggacgaca 180 gactacgctg cacccgtgaa agacagattc accatttcaa gagatgattc taaaaata tg 240 ttgtttctgc aaatggacag cctgaaaatc gaggacacag ccgtgtattt ctgtaccacg 300 gccgattttt ggagtgctta ttcttctgac tactggggcc agggaaccct ggtcaccgtc 360 tcctcaggag gtggaggttc cgggggcggg ggctccggcg gaggtggatc agatattgtg 420 atgactcagt ctccactctc cctgcccgtc acccctggag agccggcctc catctcctgc 480 aggtctagtc agagcctcct gcatagtaat gggtacaact atttggattg gtacctacag 540 aagccagggc agtctccaca actcctgatc tatttgggtt ctaatcgggc ctccggggtc 600 cctgacaggt tcagtggcag tggatcaggc acagatttta cactgaaaat cagcagaatg 660 gaggctgagg atgttggggt ttattactgc atgcaaggtc tacaaactcc gtacactttt 720 ggccagggga ccaagctgga gatcaaagga ggcggaggga gtgttttgtt ttatctggcc 780 gttggataa tgttctcgt aaatacagta ctttgggtaa caataaggaa ggaactgaag 840 agaaagaaaa aatgggatct ggaaatatca ttggacagtg gacacgaaaa aaaagtcaca 900 tcatcattgc aagaagaccg gcacttggag gaggaactga aatgtcaaga gcaaaaagaa 960 gaacaactgc aagaaggcgt acatagaaaa gaaccacagg gagcaacata g 1011 <210> 46 <211> 2669 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 46 agcctagg cc tccaaaaaag cctcctcact acttctggaa tagctcagag gcagaggcgg 60 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac 120 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat 180 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg 240 tt gctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact 300 ttccacaccg gatccaccat gggttcagct attgagcagg atgggttgca tgctggtagt 360 cccgccgcat gggtcgaacg actgtttgga tacgattggg cccaacagac tataggctgt 42 0 tccgacgctg ctgtctttcg tctttctgca caaggtcgtc cagttctgtt cgtgaaaacc 480 gacttgtccg gagccctcaa tgagttgcaa gacgaagctg cacgactgag ttggcttgcc 540 accactggtg tcccatgtgc cgcagtactt gacgtcgtca cagaggctgg tcgcgattgg 600 ttgctccttg gagaagtgcc cggccaagat cttctcagtt cccaccttgc ccctgccgaa 660 aaagtttcaa taatgg ctga cgctatgaga aggctgcaca cccttgaccc tgccacatgt 720 ccattcgatc accaagccaa acaccgaatt gaacgagcta gaacccgcat ggaagccggc 780 ctcgttgatc aagacgattt ggatgaggaa caccagggtc tcgcacccgc tgaactcttc 840 gctc gcctca aagcacgaat gccagacgga gatgacttgg tcgtaaccca cggagatgcc 900 tgccttccta acataatggt agagaatgga agatttagcg gcttcattga ttgtggacga 960 cttggagttg cagatcggta ccaagatatc gctctcgcta ccagagatat tgctgaagaa 1020 ttgggcggag aatgggctga tcggtttctc gtactctacg gaattgccgc acctgattcc 1080 caacgcattg ctttttaccg tcttctggat gagttcttct aaacgcgtcc cccctctccc 1140 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc 1200 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc 1260 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt 1320 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct 1380 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa 1440 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt 1500 tggatagttg tggaaagagt caaatggctc t cctcaagcg tattcaacaa ggggctgaag 1560 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt 1620 acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc gaaccacggg gacgtggttt 1680 tccttttgaaa aacacgattg ctcgaatcac catggtgagc aagggcgagg agctgttcac 1740 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 1800 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 1860 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgca 1920 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac tt cttcaagt ccgccatgcc 1980 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 2040 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 2100 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 2160 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 2220 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 2280 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgagcaa 2340 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 2400 cactctcggc atggacgagc tgtacaagta atcggccgct aatcagccat accacatttg 2460 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa 2520 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taat ggttac aaataaagca 2580 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 2640 ccaaactcat caatgtatct tatcatgtc 2669 <210> 47 <211> 2992 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 47 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttac ggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc t acttggcag tacatctacg 360 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg 600 atagagatct ccctatcagt gatagagatc gtcgacgttt agtgaaccgt cagatcgcct 660 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc 720 gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac gtaagtaccg 780 cctatagagt ctataggccc acccccttgg cttcttatgc atgctatact gtttttggct 840 tggggtctat acacccccgc ttcctcatgt tataggtgat ggtatagctt agcctatagg 900 tgtgggttat tgaccattat tgaccactcc cctattggtg acgatactt t ccattactaa 960 tccataacat ggctctttgc cacaactctc tttatggct atatgccaat acactgtcct 1020 tcagagactg acacggactc tgtattttta caggatgggg tctcatttat tatttacaaa 1080 ttcacatata caacaccacc gtccccagtg cccgcagttt ttattaaaca taacgtggga 1140 tctccacgcg aatctcgggt acgtgttccg gacatggtct cttctccggt agcggcggag 1200 cttctacatc cgagccctgc tcccatgcct ccagcgactc atggtcgctc ggcagctcct 1260 tgctcctaac agtggaggcc agacttaggc acagcacgat gcccaccacc accagtgtgc 1320 cgcacaaggc cgtggcggta gggtatgtgt ctgaaaatga gctc ggggag cgggcttgca 1380 ccgctgacgc atttggaaga cttaaggcag cggcagaaga agatgcaggc agctgagttg 1440 ttgtgttctg ataagagtca gaggtaactc ccgttgcggt gctgttaacg gtggagggca 1500 gtgtagtctg agcagtactc gttgctgccg cgcgcgccac cagacataat agctgacaga 1560 ctaacagact gttcctttcc atgggtcttt tctgcagaag cttatactcg agctctagat 162 0 tgggaacccg ggtctctcga attcgagatc tccaccatgc acagacctag acgtcgtgga 1680 actcgtccac ctccactggc actgctcgct gctctcctcc tggctgcacg tggtgctgat 1740 gcagaggtgc agctggtgga gtctggggga gccatagtaa agccgggg gg gtcc cataga 1800 gtctcctgtg aagcctctgg attcactttc agtaacgcct ggatgagttg ggtccgccag 1860 gctccaggga gggggctgga gtgggttggc cgtattttaa gcaagactga tggtgggacg 1920 acagactacg ctgcacccgt gaaagacaga ttcaccattt caagagatga ttctaaaaat 1980 atgttgtttc tgcaaatgga cagcctgaaa atcgaggaca cagccgtgta tttctgtacc 2040 ac ggccgatt tttggagtgc ttattcttct gactactggg gccagggaac cctggtcacc 2100 gtctcctcag gaggtggagg ttccgggggc gggggctccg gcggaggtgg atcagatatt 2160 gtgatgactc agtctccact ctccctgccc gtcacccctg gagagccggc ctccatct cc 2220 tgcaggtcta gtcagagcct cctgcatagt aatgggtaca actatttgga ttggtaccta 2280 cagaagccag ggcagtctcc acaactcctg atctatttgg gttctaatcg ggcctccggg 2340 gtccctgaca ggttcagtgg cagtggatca ggcacagatt ttacactgaa aatcagcaga 2400 atggaggctg aggatgttgg ggtttattac tgcatgcaag gtctacaaac tccgtacact 2460 tttggccagg ggaccaag ct ggagatcaaa ggaggcggag ggaggtgtttt gttttatctg 2520 gccgttggga taatgtttct cgtaaataca gtactttggg taacaataag gaaggaactg 2580 aagagaaaga aaaaatggga tctggaaata tcattggaca gtggacga aaaaaaagtc 2640 acatcatcat tgcaagaaga ccggcacttg gaggaggaac tgaaatgtca agagcaaaaa 2700 gaagaacaac tgcaagaagg cgtacataga aaagaaccac agggagcaac ataggcggcc 2760 gctaatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct 2820 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc 2880 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 2940 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tc 2992 <210> 48 <211> 5765 <212> DNA <213> Artificial sequence <220 > <223> synthetic <400> 48 acaacttcgt atagcataca ttatacgaag ttatggtacc aagcctaggc ctccaaaaaa 60 gcctcctcac tacttctgga atagctcaga ggcagaggcg gcctcggcct ctgcataaat 120 aaaaaaaatt agtcagccat ggggcggaga atgggc ggaa ctgggcggag ttaggggcgg 180 gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca tgctttgcat 240 acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg 300 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc ggatccacca 360 tgggttcagc tattgagcag gatgggttgc atgctggtag tcccgccgca tgggtcgaac 420 gactgtttgg atacgattgg gcccaacaga ctataggctg ttccgacgct gctgtctttc 480 gtctttctgc acaaggtcgt ccagttctgt tcgtgaaaac cgact c ccctgccga aaaagtttca ataatggctg 720 acgctatgag aaggctgcac acccttgacc ctgccacatg tccattcgat caccaagcca 780 aacaccgaat tgaacgagct agaacccgca tggaagccgg cctcgttgat caagacgatt 840 tggatgagga acaccagggt ctcgcacccg ctgaactctt cgctcgcctc aaagcacgaa 900 tgccagacgg agatgacttg gtcgtaaccc acggagatgc ctgccttcc t aacataatgg 960 tagagaatgg aagatttagc ggcttcattg attgtggacg acttggagtt gcagatcggt 1020 accaagatat cgctctcgct accagagata ttgctgaaga attgggcgga gaatgggctg 1080 atcggtttct cgtactctac ggaattgccg cacctgattc ccaacgcatt g ctttttacc 1140 gtcttctgga tgagttcttc taaacgcgtc ccccctctcc ctcccccccc cctaacgtta 1200 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca 1260 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca 1320 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tct gttgaat gtcgtgaagg 1380 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc 1440 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata 1500 cacctgcaaa ggcggcacaa ccccagt gcc acgttgtgag ttggatagtt gtggaaagag 1560 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc 1620 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt 1680 taaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatt 1740 gctcgaatca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 1800 gtcga gctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 1860 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 1920 ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgc tacccc 1980 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 2040 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 2100 ggcgacccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 2160 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 2220 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 2280 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 2340 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc 2400 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 2460 ctgtacaagt aatcggccgc taatcagcca taccacattt gtagaggttt tacttgcttt 2520 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt 2580 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 2640 aaataaagca tttttttc ac tgcattctag ttgtggtttg tccaaactca tcaatgtatc 2700 ttatcatgtc ggcgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 2760 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc 2820 cg cctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 2880 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggaggtattta cggtaaactg 2940 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 3000 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 3060 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt g atgcggttt tggcagtaca 3120 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 3180 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 3240 ccgccccatt gacgcaaat g ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 3300 ctctccctat cagtgataga gatctcccta tcagtgatag agatcgtcga cgtttagtga 3360 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg 3420 accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga 3480 gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttct tatgcatgct 3540 atactgtttt tggcttgggg tctatacacc cccgcttcct catgttatag gtgatggtat 3600 agcttagcct agttaggtgg gttattgacc attattgacc actcccctat tggtgacgat 366 0 actttccatt actaatccat aacatggctc tttgccacaa ctctctttat tggctatatg 3720 ccaatacact gtccttcaga gactgacacg gactctgtat ttttacagga tggggtctca 3780 tttattattt acaaattcac atatacaaca ccaccgtccc cagtgcccgc agtttttatt 3840 aaacataacg tgggatctcc acgcgaatct cgggtacgtg ttccggacat ggtctcttct 39 00 ccggtagcgg cggagcttct acatccgagc cctgctccca tgcctccagc gactcatggt 3960 cgctcggcag ctccttgctc ctaacagtgg aggccagact taggcacagc acgatgccca 4020 ccaccaccag tgtgccgcac aaggccgtgg cggtagggta tgtgtctgaa aatgagct cg 4080 gggagcgggc ttgcaccgct gacgcatttg gaagacttaa ggcagcggca gaagaagatg 4140 caggcagctg agttgttgtg ttctgataag agtcagaggt aactcccgtt gcggtgctgt 4200 taacggtgga gggcagtgta gtctgagcag tactcgttgc tgccgcgcgc gccaccagac 4260 ataatagctg acagactaac agactgttcc tttccatggg tcttttctgc agtcaccgtc 43 20 cttgacacga agcttatact cgagctctag attgggaacc cgggtctctc gaattcgaga 4380 tctccaccat gcacagacct agacgtcgtg gaactcgtcc acctccactg gcactgctcg 4440 ctgctctcct cctggctgca cgtggtgctg atgcagaggt gcagctggtg gagtct gggg 4500 gagccatagt aaagccgggg gggtcccata gagtctcctg tgaagcctct ggattcactt 4560 tcagtaacgc ctggatgagt tgggtccgcc aggctccagg gagggggctg gagtgggttg 4620 gccgtatttt aagcaagact gatggtggga cgacagacta cgctgcaccc gtgaaagaca 4680 gattcaccat ttcaagagat gattctaaaa atatgttgtt tctgcaaatg gacagcctga 4740 aaatcgagga cacagccgtg tatttctgta ccacggccga tttttggagt gcttattctt 4800 ctgactactg gggccaggga accctggtca ccgtctcctc aggaggtgga ggttccgggg 4860 gcgggggctc cggcggaggt ggatcagata ttgtgatgac tcagtctcca ctctccctgc 4920 cc gtcacccc tggagagccg gcctccatct cctgcaggtc tagtcagagc ctcctgcata 4980 gtaatgggta caactatttg gattggtacc tacagaagcc agggcagtct ccacaactcc 5040 tgatctattt gggttctaat cgggcctccg gggtccctga caggttcagt ggcagtggat 5100 caggcacaga ttttacactg aaaatcagca gaatggaggc tgaggatgtt ggggttatt 5160 actgcatgca aggtctacaacc actgtaca cttttggcca ggggaccaag ctggagatca 5220 aaggaggcgg agggagtgtt ttgttttatc tggccgttgg gataatgttt ctcgtaaata 5280 cagtactttg ggtaacaata aggaaggaac tgaagagaaa gaaaaaatgg gatctggaaa 5340 tatcattgga ca gtggacac gaaaaaaaag tcacatcatc attgcaagaa gaccggcact 5400 tggaggagga actgaaatgt caagagcaaa aagaagaaca actgcaagaa ggcgtacata 5460 gaaaagaacc acagggagca acataggcgg ccgctaatca gccataccac atttgtagag 5520 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat 5580 gcaattgttg ttgttaactt gtttattgca gcttataatg gtta caaata aagcaatagc 5640 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 5700 ctcatcaatg tatcttatca tgtctaccgg tataacttcg tataatgtat actatacgaa 5760 gttag 5765 <210> 49 <211> 660 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 49 gacatcgtga tgacccagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctagtca gagcctcctg catagtaatg ggtacaacta tttggattgg 120 tacctacaga agccagggca gt ctccacaa ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagaatgg aggctgagga tgttggggtt tattactgca tgcaaggtct acaaactccg 300 tacacttttg gccaggggac caagctggag atcaaacgag ctgatgctgc accaactgta 360 tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 420 ttgaacaact tctaccccaa agacatcaat gtcaagtgga agatt gatgg cagtgaacga 480 caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 540 agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag 600 gccactcaca agacatcaac ttcacccatt gtcaagag ct tcaacagggg agagtgttga 660 <210> 50 <211 > 1359 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 50 gaggtgcagc tggtggagtc tgggggagcc atagtaaagc cgggggggtc ccatagagtc 60 tcctgtgaag cctctggatt cactttcagt aacgcctgga tgagttgggt ccgccaggct 1 20 ccagggaggg ggctggagtg ggttggccgt attttaagca agactgatgg tgggacgaca 180 gactacgctg cacccgtgaa agacagattc accatttcaa gagatgattc taaaaatatg 240 ttgtttctgc aaatggacag cctgaaaatc gaggacacag ccgtgtattt ctgtaccacg 300 gccgattttt ggagtgctta ttcttctgac tactggggcc agggaaccct ggtcaccgtc 360 tcctcagcca aaacaacagc cccatcggtc tatccactgg cccctgtgg tggagataca 420 actggctcct cggtgactct aggatgcctg gtcaagggtt atttccctga gccagtgacc 480 ttgacctgga actctggatc cctgtccagt ggtgtgcaca ccttcccagc tgtcctgcag 540 tctgacctct acaccctcag cagctcagt actggtaacct cgagca cctg gcccagccag 600 tccatcacct gcaatgtggc ccacccggca agcagcacca aggtggacaa gaaaattgag 660 cccagagggc ccacaatcaa gccctgtcct ccatgcaaat gcccagcacc taacctcttg 720 ggtggaccat ccgtcttcat cttccctcca aagatcaagg atgtactcat gatctccctg 780 agccccatag tcacatgtgt ggtggtggat gtgagcgagg atgacccaga tgtccagatc 840 agctggttt g tgaacaacgt ggaagtacac acagctcaga cacaaaccca tagagaggat 900 tacaacagta ctctccgggt ggtcagtgcc ctccccatcc agcaccagga ctggatgagt 960 ggcaaggagt tcaaatgcaa ggtcaacaac aaagacctcc cagcgcccat cgagagaacc 1020 atctcaaaac cca aagggtc agtaagagct ccacaggtat atgtcttgcc tccaccagaa 1080 gaagagatga ctaagaaaca ggtcactctg acctgcatgg tcacagactt catgcctgaa 1140 gacatttacg tggagtggac caacaacggg aaaacagagc taaactacaa gaacactgaa 1200 ccagtcctgg actctgatgg ttcttacttc atgtacagca agctgagagt ggaaaagaag 1260 aactgggtgg aaagaa atag ctactcctgt tcagtggtcc acgagggtct gcacaatcac 1320 cacacgacta agagcttctc ccggactccg ggtaaatga 1359 <210> 51 <211> 1011 <212> DNA <213> Artificial sequence <220 > <223> synthetic <400> 51 gaggtgcagc tggtggagtc tgggggagcc atagtaaagc cgggggggtc ccatagagtc 60 tcctgtgaag cctctggatt cactttcagt aacgcctgga tgagttgggt ccgccaggct 120 ccagggaggg ggctggagtg ggttggccgt at tttaagca agactgatgg tgggacgaca 180 gactacgctg cacccgtgaa agacagattc accatttcaa gagatgattc taaaaatatg 240 ttgtttctgc aaatggacag cctgaaaatc gaggacacag ccgtgtattt ctgtaccacg 300 gccgattttt ggagtgctta ttct tctgac tactggggcc agggaaccct ggtcaccgtc 360 tcctcaggag gtggaggttc cgggggcggg ggctccggcg gaggtggatc agatattgg 420 atgactcagt ctccactctc cctgcccgtc acccctggag agccggcctc catctcctgc 480 aggtctagtc agagcctcct gcatagtaat gggtacaact atttggattg gtac ctacag 540 aagccagggc agtctccaca actcctgatc tatttgggtt ctaatcgggc ctccggggtc 600 cctgacaggt tcagtggcag tggatcaggc acagatttta cactgaaaat cagcagaatg 660 gaggctgagg atgttggggt ttattactgc atgcaaggtc tacaaactcc gtacactt tt 720 ggccagggga ccaagctgga gatcaaagga ggcggaggga gtgttttgtt ttatctggcc 780 gttggataa tgtttctcgt aaatacagta ctttgggtaa caataaggaa ggaactgaag 840 agaaagaaaa aatgggatct ggaaatatca ttggacagtg gacacgaaaa aaaagtcaca 900 tcatcattgc aagaagaccg gcacttggag gaggaactga aatgtcaaga gcaaaaaga a 960gaacaactgc aagaaggcgt acatagaaaa gaaccacagg gagcaacata g 1011

Claims (115)

A method for detecting and isolating cells that produce high levels of bispecific antibodies, comprising:
(a) transfecting a cell with a nucleic acid encoding a cell surface capture protein (CSCP), a fusion protein comprising an antigen-binding protein and a membrane anchor domain, wherein the cell expresses a bispecific antibody, and An enemy antibody comprises multiple subunits wherein the first site on the dual specific antibody is on the first subunit and the second site on the dual specific antibody is on the second subunit, (i) the CSCP is on the dual subunit. binds to the first site on the specific antibody to form a CSCP-bispecific antibody complex inside the host cell, (ii) the CSCP-bispecific antibody complex is transported through the host cell, and (iii) then the host cell displayed on a surface;
(b) selecting cells of (a) expressing CSCP in high yield;
(c) isolating and culturing cells expressing CSCP in high yield;
(d) detecting the bispecific antibody on the surface of the isolated and cultured cells of step (c) with a detection molecule (DM) that binds to a second site on the bispecific antibody, wherein the DM is a labeled recombinant antigen -comprising a binding protein; and
(e) isolating the cells detected in step (d) that contain the bispecific antibody detected on the surface,
At this time, the first subunit and the second subunit of the dual specific antibody
(i) a CH3 domain (Fc) having a histidine residue (95H) at position 95 according to the IMGT exon numbering system and a tyrosine residue (96Y) at position 96 according to the IMGT exon numbering system; or
(ii) a CH3 domain (Fc*) containing an arginine residue (95R) at position 95 according to the IMGT exon numbering system and a phenylalanine residue (96F) at position 96 according to the IMGT exon numbering system;
Said dual specific antibody comprises both Fc and Fc*;
The first subunit and the second subunit of the dual specific antibody are different;
if the first subunit of the bispecific antibody is Fc then the second subunit of the bispecific antibody is Fc*;
if the first subunit of the bispecific antibody is Fc* then the second subunit of the bispecific antibody is Fc;
The antigen-binding protein of CSCP and the labeled recombinant antigen-binding protein of DM are different;
The antigen-binding protein of CSCP or the labeled recombinant antigen-binding protein of DM operably binds Fc, and the antigen-binding protein of CSCP or the labeled recombinant antigen-binding protein of DM:
a. a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:15 and a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:16;
b. Heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO:27, HCDR-2 having the amino acid sequence of SEQ ID NO:28, HCDR-3 having the amino acid sequence of SEQ ID NO:29, SEQ ID light chain CDR-1 (LCDR-1) having the amino acid sequence of NO:30, and LCDR-2 having the amino acid sequence of SEQ ID NO:31;
c. heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO:27, HCDR-2 having the amino acid sequence of SEQ ID NO:28, HCDR-3 having the amino acid sequence of SEQ ID NO:29, and SEQ a light chain variable region having the amino acid sequence of ID NO:16; or
d. a ScFv fusion protein comprising the amino acid sequence of SEQ ID NO:19; or
The antigen-binding protein of CSCP or the labeled recombinant antigen-binding protein of DM operably binds Fc*, and the antigen-binding protein of CSCP or the labeled recombinant antigen-binding protein of DM:
(i) a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:38 and a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:39;
(ii) heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO:32, HCDR-2 having the amino acid sequence of SEQ ID NO:33, HCDR-3 having the amino acid sequence of SEQ ID NO:34 , light chain CDR-1 (LCDR-1) having the amino acid sequence of SEQ ID NO:35, LCDR-2 having the amino acid sequence of SEQ ID NO:36, and LCDR-3 having the amino acid sequence of SEQ ID NO:37;
(iii) a ScFv fusion protein comprising the amino acid sequence of SEQ ID NO:43; or
(iv) a heavy chain having the amino acid sequence of SEQ ID NO:40 and a light chain having the amino acid sequence of SEQ ID NO:41. A method for detecting or isolating cells stably expressing a bispecific antibody,
(a) expressing in a host cell a cell surface capture protein (CSCP) comprising an antigen-binding protein, and a bispecific antibody, wherein the bispecific antibody comprises multiple subunits and One site is on the first subunit, the second site on the bispecific antibody is on the second subunit, and (i) CSCP binds to the first site on the bispecific antibody to produce CSCP- forming a bispecific antibody complex, (ii) the CSCP-bispecific antibody complex is delivered through the host cell, and (iii) then displayed on the surface of the host cell;
(b) contacting the host cell with a detection molecule (DM), wherein the detection molecule binds to a second site on the bispecific antibody; and
(c) selecting a host cell that binds to the detection molecule,
At this time, the first subunit and the second subunit of the dual specific antibody
(i) a CH3 domain (Fc) having a histidine residue (95H) at position 95 according to the IMGT exon numbering system and a tyrosine residue (96Y) at position 96 according to the IMGT exon numbering system; or
(ii) a CH3 domain (Fc*) containing an arginine residue (95R) at position 95 according to the IMGT exon numbering system and a phenylalanine residue (96F) at position 96 according to the IMGT exon numbering system;
Said dual specific antibody comprises both Fc and Fc*;
The first subunit and the second subunit of the dual specific antibody are different;
if the first subunit of the bispecific antibody is Fc then the second subunit of the bispecific antibody is Fc*;
if the first subunit of the bispecific antibody is Fc* then the second subunit of the bispecific antibody is Fc;
The antigen-binding protein of CSCP and the labeled recombinant antigen-binding protein of DM are different;
The antigen-binding protein of CSCP or the labeled recombinant antigen-binding protein of DM operably binds Fc, and the antigen-binding protein of CSCP or the labeled recombinant antigen-binding protein of DM:
a. a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:15 and a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:16;
b. Heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO:27, HCDR-2 having the amino acid sequence of SEQ ID NO:28, HCDR-3 having the amino acid sequence of SEQ ID NO:29, SEQ ID light chain CDR-1 (LCDR-1) having the amino acid sequence of NO:30, and LCDR-2 having the amino acid sequence of SEQ ID NO:31;
c. heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO:27, HCDR-2 having the amino acid sequence of SEQ ID NO:28, HCDR-3 having the amino acid sequence of SEQ ID NO:29, and SEQ a light chain variable region having the amino acid sequence of ID NO:16; or
d. a ScFv fusion protein comprising the amino acid sequence of SEQ ID NO:19; or
The antigen-binding protein of CSCP or the labeled recombinant antigen-binding protein of DM operably binds Fc*, and the antigen-binding protein of CSCP or the labeled recombinant antigen-binding protein of DM:
(i) a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:38 and a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:39;
(ii) heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO:32, HCDR-2 having the amino acid sequence of SEQ ID NO:33, HCDR-3 having the amino acid sequence of SEQ ID NO:34 , light chain CDR-1 (LCDR-1) having the amino acid sequence of SEQ ID NO:35, LCDR-2 having the amino acid sequence of SEQ ID NO:36, and LCDR-3 having the amino acid sequence of SEQ ID NO:37;
(iii) a ScFv fusion protein comprising the amino acid sequence of SEQ ID NO:43; or
(iv) a heavy chain having the amino acid sequence of SEQ ID NO:40 and a light chain having the amino acid sequence of SEQ ID NO:41. 3. The method according to claim 1 or 2, characterized in that the first subunit of the dual specific antibody comprises a heavy chain comprising a CH3 domain (Fc) with 95H and 96F according to the IMGT exon numbering system. 3. The method according to claim 1 or 2, characterized in that the second subunit comprises a heavy chain comprising a substituted CH3 domain (Fc*) comprising 95R and 96F according to the IMGT exon numbering system. According to claim 1 or 2,
(i) the first subunit of the dual specific antibody comprises a CH3 domain (Fc) with 95H and 96Y according to the IMGT exon numbering system; and
(ii) the second subunit of the dual specific antibody comprises a CH3 domain (Fc*) comprising 95R and 96F according to the IMGT exon numbering system. 3. The method of claim 1 or 2, wherein the antigen-binding protein of CSCP binds to a human IgG1-Fc domain, a human IgG2-Fc domain, or a human IgG4-Fc domain. 3. The method according to claim 1 or 2, characterized in that the first subunit comprises a CH3 domain (Fc*) comprising 95R and 96F according to the IMGT exon numbering system. 3. The method according to claim 1 or 2, characterized in that the second subunit of the dual specific antibody comprises a CH3 domain (Fc) with 95H and 96Y according to the IMGT exon numbering system. According to claim 1 or 2,
(i) the first subunit of the dual specific antibody comprises a CH3 domain (Fc*) comprising 95R and 96F according to the IMGT exon numbering system;
(ii) the second subunit of the dual specific antibody comprises a CH3 domain (Fc) with 95H and 96Y according to the IMGT exon numbering system. delete 3. The method of claim 1 or 2, further comprising contacting the cell with a blocking molecule, wherein the blocking molecule binds CSCP not bound to the bispecific antibody, but binds to the CSCP-bispecific antibody complex. A method characterized by not doing. 12. The method of claim 11, wherein the blocking molecule is a non-human IgG or human Fc molecule. 3. The method according to claim 1 or 2, wherein the cells are detected, isolated or selected by fluorescence activated cell sorting. 3. The method according to claim 1 or 2, further comprising the step of pooling the detected and selected cells that produce the bispecific antibody. 15. The method of claim 14, further comprising culturing and expanding the pooled cells. 16. The method of claim 15, wherein the pooled cells are enriched by further selection and detection by contacting the pooled cells with a detection molecule. 17. The method of claim 16, wherein the further detection and selection of cells is by fluorescence activated cell sorting. 17. The method of claim 16, wherein cells producing the bispecific antibody are detected, selected and pooled multiple times to enrich for cells producing the high titer of the bispecific antibody. 3. The method according to claim 1 or 2, wherein the detection molecule (DM) is
a. A labeled recombinant antigen-binding protein that binds to a human IgG1-Fc domain, a human IgG2-Fc domain, or a human IgG4-Fc domain, wherein the Fc domain comprises an arginine residue at position 95 according to the IMGT exon numbering system and an arginine residue according to the IMGT exon numbering system. A labeled recombinant antigen-binding protein comprising a phenylalanine residue at position 96; or
b. A labeled recombinant antigen-binding protein that binds to a human IgG3-Fc domain, wherein the Fc domain comprises a histidine residue at position 95 according to the IMGT exon numbering system and a tyrosine residue at position 96 according to the IMGT exon numbering system. binding protein
A method comprising a. 3. The method according to claim 1 or 2, characterized in that the bispecific antibody and cell surface capture protein (CSCP) are recombinantly expressed as a fusion protein. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete